Compositions and methods of using (r)-pramipexole

ABSTRACT

Pharmaceutical compositions of (R)-pramipexole and one or more secondary therapeutic agents such as, for example, dopamine agonists, dopaminergic agonists, COMT inhibitors, MOA inhibitors, excitatory amino acid antagonists, growth factors, neurotrophic factors, antioxidants, anti-inflammatory agents, immunomodulators, anti-glutamatergics, ion channel blockers, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists, heat shock protein inducers/protein disaggregators and downregulators, monoamine oxidase type B (MOAB) inhibitors, multi-target agents, kinase inhibitors, Bcl inducers, histone deacetylase (HDAC) mediators, glial modulators, mitochondrial energy promoting agents, myostatin inhibitors, caspase inhibitors and combinations thereof or those related to mitochondrial dysfunction or increased oxidative stress are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/059,713 filed Apr. 19, 2011 entitled “Compositions and Methods ofUsing (R)-Pramipexole”, which is a U.S. national stage filing under 35U.S.C. § 371 of International Application No. PCT/US2009/054292 filedAug. 19, 2009 entitled “Compositions and Methods of Using(R)-Pramipexole”, which claims the priority benefit of U.S. ProvisionalApplication No. 61/090,094 filed Aug. 19, 2008 entitled “Compositionsand Methods of Using (R)-Pramipexole”, and U.S. Provisional ApplicationNo. 61/113,680 filed Nov. 12, 2008 entitled “Compositions and Methods ofUsing (R)-Pramipexole”, each of which are herein incorporated byreference in their entireties.

GOVERNMENT INTERESTS

Not Applicable

PARTIES TO A JOINT RESEARCH AGREEMENT

Not Applicable

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

Not Applicable

BACKGROUND

Not Applicable

BRIEF SUMMARY OF THE INVENTION

Various embodiments of the invention are directed to a multi-componenttherapeutic including a first component comprising a therapeuticallyeffective amount of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine and a secondcomponent comprising a therapeutically effective amount of one or moresecondary therapeutic agents. In some embodiments, the second componentmay be dopamine agonists, dopaminergic agonists, COMT inhibitors, MOAinhibitors, excitatory amino acid antagonists, growth factors,neurotrophic factors, antioxidants, anti-inflammatory agents,immunomodulators, anti-glutamatergics, ion channel blockers,α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptorantagonists, heat shock protein inducers/protein disaggregators anddownregulators, monoamine oxidase type B (MOAB) inhibitors, multi-targetagents, kinase inhibitors, Bcl protein inducers, histone deacetylase(HDAC) mediators, glial modulators, mitochondrial energy promotingagents, myostatin inhibitors, caspase inhibitors and combinationsthereof.

Various other embodiments of the invention are directed to a method oftreating a neurodegenerative disease in a patient including the steps ofadministering a first component comprising a therapeutically effectiveamount of (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine tothe patient and administering adjunctively a second component comprisinga therapeutically effective amount of one or more secondary therapeuticagents to the patient. In some embodiments, the second component may beselected from dopamine agonists, dopaminergic agonists, COMT inhibitors,MOA inhibitors, excitatory amino acid antagonists, growth factors,neurotrophic factors, antioxidants, anti-inflammatory agents,immunomodulators, anti-glutamatergics, ion channel blockers,α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptorantagonists, heat shock protein inducers/protein disaggregators anddownregulators, monoamine oxidase type B (MOAB) inhibitors, multi-targetagents, kinase inhibitors, Bcl protein inducers, histone deacetylase(HDAC) mediators, glial modulators, mitochondrial energy promotingagents, myostatin inhibitors, caspase inhibitors and combinationsthereof. In other embodiments, the neurodegenerative disease may beselected from Huntington's Chorea, metabolically induced neurologicaldamage, senile dementia of Alzheimer's type, age associated cognitivedysfunction, vascular dementia, multi-infarct dementia, Lewy bodydementia, neurodegenerative dementia, neurodegenerative movementdisorder, ataxia, Friedreich's ataxia, multiple sclerosis, spinalmuscular atrophy, primary lateral sclerosis, seizure disorders, motorneuron disorder or disease, inflammatory demyelinating disorder,Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis,hepatic encephalopathy, and chronic encephalitis.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the nature and advantages of the presentinvention, reference should be made to the following detaileddescription taken in connection with the accompanying drawings, inwhich:

FIG. 1 depicts the mean plasma (R)-pramipexole concentrations after oraladministration of single 50 mg, 150 mg, and 300 mg doses to healthyvolunteers under fasted conditions.

FIG. 2 depicts mean plasma (R)-pramipexole concentrations after oraladministration of single 150 mg doses to healthy volunteers under fastedand fed conditions.

FIG. 3 depicts mean plasma (R)-pramipexole concentrations on Days 1 and7 during oral administration of 50 mg and 100 mg doses on Day 1, Q12H onDays 3 through 6, and a single dose on Day 7 to healthy volunteers underfasted conditions.

FIG. 4 depicts an exposure (AUC) vs. dose (mg/m²) for male and femalerats and humans (both genders).

FIG. 5 depicts mean exposure (AUC) vs. dose (mg/m²) for male and femaleminipigs and humans (both genders).

DETAILED DESCRIPTION

Before the compositions and methods provided herein are described, it isto be understood that this invention is not limited to the particularprocesses, compositions, or methodologies described, as these may vary.It is also to be understood that the terminology used in the descriptionis for the purpose of describing the particular versions or embodimentsonly, and is not intended to limit the scope of the present inventionwhich will be limited only by the appended claims. All publicationsmentioned herein are incorporated by reference in their entirety to theextent to support the present invention.

It must be noted that, as used herein, and in the appended claims, thesingular forms “a”, “an” and “the” include plural reference unless thecontext clearly dictates otherwise. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art. Although anymethods similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the present invention, thepreferred methods are now described. All publications and referencesmentioned herein are incorporated by reference. Nothing herein is to beconstrued as an admission that the invention is not entitled to antedatesuch disclosure by virtue of prior invention.

As used herein, the term “about” means plus or minus 10% of thenumerical value of the number with which it is being used. Therefore,about 50% means in the range of 45%-55%.

“Optional” or “optionally” may be taken to mean that the subsequentlydescribed structure, event or circumstance may or may not occur, andthat the description includes instances where the event occurs andinstances where it does not.

“Administering” when used in conjunction with a therapeutic means toadminister a therapeutic directly into or onto a target tissue or toadminister a therapeutic to a patient whereby the therapeutic positivelyimpacts the tissue to which it is targeted. “Administering” acomposition may be accomplished by oral administration, injection,infusion, absorption or by any method in combination with other knowntechniques. Such combination techniques include heating, radiation andultrasound.

The term “target”, as used herein, refers to the material for whicheither deactivation, rupture, disruption or destruction or preservation,maintenance, restoration or improvement of function or state is desired.For example, diseased cells, pathogens, or infectious material may beconsidered undesirable material in a diseased subject and may be atarget for therapy.

Generally speaking, the term “tissue” refers to any aggregation ofsimilarly specialized cells which are united in the performance of aparticular function.

The term “improves” is used to convey that the present invention changeseither the appearance, form, characteristics and/or physical attributesof the tissue to which it is being provided, applied or administered.“Improves” may also refer to the overall physical state of an individualto whom an active agent has been administered. For example, the overallphysical state of an individual may “improve” if one or more symptoms ofa neurodegenerative disorder are alleviated by administration of anactive agent.

As used herein, the term “therapeutic” means an agent utilized to treat,combat, ameliorate or prevent an unwanted condition or disease of apatient.

The terms “therapeutically effective amount” or “therapeutic dose” asused herein are interchangeable and may refer to the amount of an activeagent or pharmaceutical compound or composition that elicits abiological or medicinal response in a tissue, system, animal, individualor human that is being sought by a researcher, veterinarian, medicaldoctor or other clinician. A biological or medicinal response mayinclude, for example, one or more of the following: (1) preventing adisease, condition or disorder in an individual that may be predisposedto the disease, condition or disorder but does not yet experience ordisplay pathology or symptoms of the disease, condition or disorder, (2)inhibiting a disease, condition or disorder in an individual that isexperiencing or displaying the pathology or symptoms of the disease,condition or disorder or arresting further development of the pathologyand/or symptoms of the disease, condition or disorder, and (3)ameliorating a disease, condition or disorder in an individual that isexperiencing or exhibiting the pathology or symptoms of the disease,condition or disorder or reversing the pathology and/or symptomsexperienced or exhibited by the individual.

The term “unit dose” as used herein may be taken to indicate a discreteamount of the therapeutic composition which comprises a predeterminedamount of the active compound. The amount of the active ingredient isgenerally equal to the dosage of the active ingredient which may beadministered once per day, or may be administered several times a day(e.g. the unit dose is a fraction of the desired daily dose). The unitdose may also be taken to indicate the total daily dose, which may beadministered once per day or may be administered as a convenientfraction of such a dose (e.g. the unit dose is the total daily dosewhich may be given in fractional increments, such as, for example,one-half or one-third the dosage).

As used herein, the term “neuroprotectant” refers to any agent that mayprevent, ameliorate or slow the progression of neuronal degenerationand/or neuronal cell death.

The term “treating” may be taken to mean prophylaxis of a specificdisorder, disease or condition, alleviation of the symptoms associatedwith a specific disorder, disease or condition and/or prevention of thesymptoms associated with a specific disorder, disease or condition.

The term “patient” generally refers to any living organism to which tocompounds described herein are administered and may include, but is notlimited to, any non-human mammal, primate or human. Such “patients” mayor my not be exhibiting the signs, symptoms or pathology of theparticular diseased state.

As used herein, the terms “enantiomers”, “stereoisomers” and “opticalisomers” may be used interchangeably and refer to molecules whichcontain an asymmetric or chiral center and are mirror images of oneanother. Further, the terms “enantiomers”, “stereoisomers” or “opticalisomers” describe a molecule which, in a given configuration, cannot besuperimposed on its mirror image.

As used herein, the terms “optically pure” or “entantiomerically pure”may be taken to indicate that a composition contains at least 99.95% ofa single optical isomer of a compound. The term “entantiomericallyenriched” may be taken to indicate that at least 51% of a composition isa single optical isomer or enantiomer. The term “entantiomericenrichment” as used herein refers to an increase in the amount of oneentantiomer as compared to the other. A “racemic” mixture is a mixtureof equal amounts of (6R) and (6S) enantiomers of a chiral molecule.

Throughout this disclosure, the word “pramipexole” or “(S)-pramipexole”will refer to (6S) enantiomer of2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole unless otherwisespecified, and(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine will bereferred to as “(R)-pramipexole” or “RPPX.”

The term “pharmaceutical composition” shall mean a composition includingat least one active ingredient, whereby the composition is amenable toinvestigation for a specified, efficacious outcome in a mammal (forexample, without limitation, a human). Those of ordinary skill in theart will understand and appreciate the techniques appropriate fordetermining whether an active ingredient has a desired efficaciousoutcome based upon the needs of the artisan. A pharmaceuticalcomposition may, for example, contain pramipexole or a pharmaceuticallyacceptable salt of pramipexole as the active ingredient. Alternatively,a pharmaceutical composition may contain (R)-pramipexole or apharmaceutically acceptable salt of (R)-pramipexole as the activeingredient.

For the purposes of this disclosure, a “salt” is any acid addition salt,preferably a pharmaceutically acceptable acid addition salt, includingbut not limited to, halogenic acid salts such as hydrobromic,hydrochloric, hydrofluoric and hydroiodic acid salt; an inorganic acidsalt such as, for example, nitric, perchloric, sulfuric and phosphoricacid salt; an organic acid salt such as, for example, sulfonic acidsalts (methanesulfonic, trifluoromethan sulfonic, ethanesulfonic,benzenesulfonic or p-toluenesulfonic), acetic, malic, fumaric, succinic,citric, benzoic, gluconic, lactic, mandelic, mucic, pamoic, pantothenic,oxalic and maleic acid salts; and an amino acid salt such as aspartic orglutamic acid salt. The acid addition salt may be a mono- or di-acidaddition salt, such as a di-hydrohalogenic, di-sulfuric, di-phosphoricor di-organic acid salt. In all cases, the acid addition salt is used asan achiral reagent which is not selected on the basis of any expected orknown preference for interaction with or precipitation of a specificoptical isomer of the products of this disclosure.

“Pharmaceutically acceptable salt” is meant to indicate those saltswhich are, within the scope of sound medical judgment, suitable for usein contact with the tissues of a patient without undue toxicity,irritation, allergic response and the like, and are commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable salts arewell known in the art. For example, Berge et al. (1977) J. Pharm.Sciences, Vol 6. 1-19, describes pharmaceutically acceptable salts indetail.

As used herein, the term “comparative binding affinity ratio” refers tothe binding affinity at the D₂ or D₃ dopamine receptors (IC₅₀ value) of(R)-pramipexole divided by the binding affinity at the D₂ or D₃ dopaminereceptors (IC₅₀ value) of (S)-pramipexole. In some embodiments, thecomparative binding affinity ratio refers to the ratio of the IC₅₀values at the D₂ receptor. In some embodiments, the comparative bindingaffinity ratio refers to the ratio of the IC₅₀ values at the D₃receptor.

As used herein, the term “comparative ratio” refers one of thefollowing: 1) the ratio of the IC₅₀ values at the D₂ or D₃ receptors for(R)-pramipexole to (S)-pramipexole; 2); the ratio of MTD amounts for(R)-pramipexole to (S)-pramipexole; or 3) the ratio of NOAEL doseamounts for (R)-pramipexole to (S)-pramipexole.

As used herein, the term “daily dose amount” refers to the amount ofpramipexole per day that is administered or prescribed to a patient.This amount can be administered in multiple unit doses or in a singleunit dose, in a single time during the day or at multiple times duringthe day.

As used herein, the term “dopaminergic activity equivalent” (DAE) refersto the measure of activity at the dopamine receptors which is equivalentto the activity of 1 mg of (S)-pramipexole at the dopamine receptors.

A “dose amount” as used herein, is generally equal to the dosage of theactive ingredient which may be administered once per day, or may beadministered several times a day (e.g. the unit dose is a fraction ofthe desired daily dose). For example, a non-effective dose amount of 0.5mg/day of (S)-pramipexole may be administered as 1 dose of 0.5 mg, 2doses of 0.25 mg each or 4 doses of 0.125 mg. The term “unit dose” asused herein may be taken to indicate a discrete amount of thetherapeutic composition which comprises a predetermined amount of theactive compound. The amount of the active ingredient is generally equalto the dosage of the active ingredient which may be administered onceper day, or may be administered several times a day (e.g. the unit doseis a fraction of the desired daily dose). The unit dose may also betaken to indicate the total daily dose, which may be administered onceper day or may be administered as a convenient fraction of such a dose(e.g. the unit dose is the total daily dose which may be given infractional increments, such as, for example, one-half or one-third thedosage).

As used herein, the terms “enantiomers”, “stereoisomers” and “opticalisomers” may be used interchangeably, and refer to molecules whichcontain an asymmetric or chiral center and are non-superimposable mirrorimages of one another. As used herein, the term “chirally pure” or“enantiomerically pure” may be taken to indicate that the compoundcontains at least 99.95% of a single optical isomer. The term“enantiomerically enriched”, unless a number is mentioned, may be takento indicate that at least 51% of the material is a single enantiomer.The term “enantiomeric enrichment” as used herein refers to an increasein the amount of one enantiomer as compared to the other. A “racemic”mixture is a mixture of equal amounts of (R)- and (S)-enantiomers of achiral molecule.

As used herein, a “kit” refers to one or more pharmaceuticalcompositions and instructions for administration or prescription of theone or more compositions. The instructions may consist of productinsert, instructions on a package of one or more pharmaceuticalcompositions, or any other instruction.

As used herein, the term “Mirapex®” refers to tablets containing(S)-pramipexole dihydrochloride, which has the chemical name,(S)-2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazoledihydrochloride monohydrate.

As used herein, the term “naïve patient” refers to a patient that hasnot previously received pramipexole treatment (either (R)-pramipexole or(S)-pramipexole) or who has not received a titration regimen ofpramipexole previous to receiving a starting dose of pramipexole.

As used herein, the term “starting daily dose amount” refers to theamount of pramipexole per day that is administered or prescribed to apatient beginning pramipexole treatment, who has not previously beensubjected to a titration regimen of pramipexole. This amount can beadministered in multiple unit doses or in a single unit dose, in asingle time during the day or at multiple times during the day.

By “adjunctive administration” or “adjunctively” is meant simultaneousadministration of more than one compound, in the same dosage form,simultaneous administration in separate dosage forms, and separateadministration of the more than one compound. For example, secondarypharmaceutical compositions such as a dopaminergic agonists and/or anantioxidant may be adjunctively administered with (R)-pramipexole wherethe (R)-pramipexole, the dopaminergic agonists, and the antioxidant areall in separate dosage forms such as 3 individual tablets, or adopaminergic agonist and/or an antioxidant my be adjunctivelyadministered with (R)-pramipexole in a single dosage form such as asingle tablet. As such, each individual pharmaceutical composition mayfurther include one or more pharmaceutically acceptable excipients orcarriers.

The term “trituration” may be taken to indicate a method of solidifyinga chemical compound. Trituration involves agitating the compound bystirring, beating or a method of the like until the chemical compoundforms a crystalline solid or precipitate. This solid may act to seed theremaining chemical compound in solution, causing it to precipitate orcrystallize from solution.

(R)-pramipexole is an enantiomer of the active pharmaceutical ingredientof the approved Parkinson's disease (PD) and restless legs syndrome(RLS) treatment Mirapex (pramipexole; (S)-pramipexole). Mirapex® is ahigh-affinity (low nM IC₅₀) agonist at human and rodent recombinantdopamine D₂ and D₃ receptors, a property that is the pharmacologicalbasis of its efficacy in these disorders. Both the (R)- and the(S)-enantiomers have been shown preclinically to possess neuroprotectiveproperties that are independent of dopamine receptor affinity.

Neuroprotective properties of (S)-pramipexole have been recognized aspotentially useful for the treatment of neurodegenerative disorders, butclinical experience with the drug for treatment of dopamine deficiencydisorders, such as PD, have shown that dosing is limited bothtemporally, by the need for prolonged dose titration, and absolutely, interms of maximum tolerated dose (MTD), due to dopamine agonist-relatedside effects. These dosing limitations are typical for dopamine receptoragonists of this class:

The maximum allowable single starting dose for Mirapex® is 0.125 mg,given three times a day (t.i.d.); and the maximum allowable dose forMirapex is 1.5 mg t.i.d., providing a maximum daily dose of 4.5 mg ofMirapex® after 7-8 weeks of titration.

While these dose levels of Mirapex® are useful for treatment of thesigns and symptoms of PD and RLS, in neuroprotective assays the potencyof (S)-pramipexole as a neuroprotective is approximately 1000-fold lowerthan its potency as a dopamine agonist. This suggests thetherapeutically useful neuroprotective doses cannot be reached usingthis enantiomer.

(R)-pramipexole possesses similar neuroprotective potency, but loweraffinity for dopamine receptors. Accordingly, it has been advanced as apotentially more useful compound for treatment of neurodegenerativedisorders. However, previously reported dopamine receptor affinitydifference for the (R)-pramipexole compared to (S)-pramipexole wouldstill impose clinically important dose limitations and would stillrequire dose-titration and dose-limitations to avoid dopamine-relatedside effects. In previous reports utilizing (R)-pramipexole inamyotrophic lateral sclerosis (ALS), a rapidly progressing fatalneurodegenerative disorder, dosing of (R)-pramipexole was suggested tobe limited and to require significant dose-titration in animalexperiments. The assumed requirement for dose-titration-specifically,the requirement to start dosing at very low doses and increase the doseto a final therapeutically effective dose level over 7-8 weeks-severelylimits the usefulness of the neuroprotective potential of the(R)-pramipexole enantiomer. Additionally, the assumed MTD would severelylimit the timely exploitation of the neuroprotective potential of the(R)-pramipexole enantiomer.

Various embodiments of the invention presented herein are directed to amulti-component system including (R)-pramipexole and one or moresecondary agents, pharmaceutical compositions including (R)-pramipexoleand one or more secondary agents, and methods for treating a disease ina subject including the steps of administering (R)-pramipexole and oneor more secondary agents. The components of the multi-component systemmay be administered individually or in combined into a single dosageformula. Therefore, some embodiments of the invention are directed to apharmaceutical compositions including (R)-pramipexole and one or moresecondary agents and a pharmaceutically acceptable excipient or carrierand methods for using such pharmaceutical compositions.

The secondary agents of embodiments may be any agent that when combinedwith (R)-pramipexole produces a beneficial effect. For example, in someembodiments, the secondary agent may include one or more dopaminergicagonists, catechol-O-methyl transferase (COMT) inhibitors, monoamineoxidase (MOA) inhibitors, excitatory amino acid antagonists andcombinations thereof. In other embodiments, the secondary agent mayinclude growth factors, neurotrophic factors, antioxidants,anti-inflammatory agents, immunomodulators, anti-glutamatergics, ionchannel blockers, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid(AMPA) receptor antagonists, heat shock protein inducers/proteindisaggregators and downregulators, monoamine oxidase type B (MOAB)inhibitors, multi-target agents, kinase inhibitors, B-cell lymphoma(Bcl) inducers, histone deacetylase (HDAC) inhibitors, glial modulators,mitochondrial energy promoting agents, myostatin inhibitors, caspaseinhibitors and combinations thereof. Embodiments of the invention arenot limited to any particular agent encompassed by the classes of agentsdescribed above, and any agent that falls within any of these categoriesmay be utilized in embodiments of the invention. Non-limiting examples,of such agents are provided for clarity.

For example, in some embodiments, exemplary dopamine agonists mayinclude, but are not limited to, apomorphine, carbidopa/levodopa,bromocriptine, lisuride, cabergoline and piribedel, and in particularembodiments, the dopamine agonists may be D2/D3 agonists such as, butnor limited to pramipexole((6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine) (e.g.,Mirapex®), ropinirole (e.g., Requip®), carbidopa, levodopa, entacapone(e.g., COMtan®), carbidopa/levodopa (e.g., Sinemet®),carbidopa/levodopa/entacapone (e.g. Stalevo®), selegiline (e.g.,Eldpryyl®), rotigotine (e.g., Neupro®), rasagaline (e.g., Azilect®),apomorphine (e.g., Apokyn®), bromocriptine (e.g., Parlodel®), amantadine(e.g., Symmetrel®) paliroden, xaliproden, talampanel and combinationsthereof. Without wishing to be bound by theory, in such embodiments,(R)-pramipexole may exert a neuroprotective effect while a D2/D3agonists may activate dopamine receptors. In further embodiments,exemplary dopaminergic agonists may include, but are not limited to,ropinirole, rotigotine, pergolide, amantadine. In other embodiments,exemplary COMT inhibitors may include, but are not limited to,entacapone and tolcapone. In still other embodiments, exemplary MOAinhibitors may include, but are not limited to, selegiline, rasagilinemoclobemide, isocarboxazid, phenelzine, tranylcypromine, nialamide,iproniazid, iproclozide, toloxatone, linezolid, dextroamphetamine, EVT302 (Evotec, Inc.), Ro 19-6491 (Hoffman-La Roche, Inc.), Ro 19-6327(Hoffman-La Roche, Inc.), deprenyl, pargyline and ladostigil (TV-3326),and in yet other embodiments, exemplary excitatory amino acidantagonists may include, but are not limited to, talampanel.

In further embodiments, exemplary growth factors and neurotrophicfactors may include, but are not limited to, insulin-like growthfactor-1 (IGF-1), IGF-1 AAV, IPLEX, glial cell line-derived neurotrophicfactor (GDNF), hepatocyte growth factor (HGF), and granulocyte colonystimulating factor(G-CSF). In some embodiments, exemplary antioxidants,anti-inflammatories, and immunomodulators may include, but are notlimited to, AEOL 10150, cefriaxone, celastrol, coenzyme Q10, copaxone,cox-2 inhibitors (including nimesulide), cyclosporin, ebselen, edaravone(radicut), promethazine, tamoxifen, thalidomide, vitamin E and VP025,and in other embodiments, exemplary AMPA receptor antagonists mayinclude, but are not limited to,1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide(NBQX) and talampanel. In still other embodiments, exemplary heat shockprotein inducers/protein disaggregators and downregulators include, butare not limited to, arimoclomol, ISIS 333611, lithium, misfolded SOD-1antibodies, rhHSP70, TDP-43 antagonists and trehalose, and in yet otherembodiments, exemplary MOAB inhibitors may include, but are not limitedto rasagiline [R(+)N-propargyl-1-aminoindan].

In particular embodiments, exemplary multi-target agents may include,but are not limited to, 4-[2(aminomethyl)-1,3-thiazol-4-yl]-2,6di-tert-butylphenol, and in some embodiments, exemplary kinaseinhibitors may include, but are not limited to, olomoucine,quinolin-2(1H)-one derivatives, roscovitine, tamoxifen and combinationsthereof. In other embodiments, exemplary Bcl inducers include, but arenot limited to, ginsenoside Rb1 and Rg1, G3139, oblimersen andcombinations thereof, and in further embodiments, exemplary HDACmediators may include, but are not limited to, phenylbutyrate,scriptaid, valproic acid and combinations thereof. In still otherembodiments, exemplary glial modulators include, but are not limited to,ONO-2506, and in yet other embodiments, exemplary mitochondrial energypromoter agents may include, but are not limited to, resveratrol,creatine, erythropoietin, cholest-4-en-3-One, and oxime (TRO-19622). Instill further embodiments, exemplary myostatin inhibitors may include,but are not limited to, ACE-031, MYO-029 and combinations thereof, andin certain embodiments, exemplary caspase inhibitors may include, butare not limited to, ESPA-1002, IDN-6556, pralnacasan and combinationsthereof.

Any of the secondary agents described above may be useful in embodimentsof the invention. However, in particular embodiments, the secondaryagent may be a dopamine agonist. For example, in one exemplaryembodiment, the dopamine agonist may be ropinirole (Requip®), and inanother exemplary embodiment, the dopamine agonist may becarbidopa/levodopa (Sinemet®). In other particular embodiments, thesecondary agent may be an anti-glutamatergic. For example, in oneexemplary embodiment, the secondary agent may be riluzole (Rilutek®). Inanother particular embodiments, the secondary agent may be an excitatoryamino acid. For example in one exemplary embodiment, the secondary agentmay be talampanel. In still other particular embodiments, the secondaryagent may be a growth factor. For example, in one exemplary embodiment,the secondary agent may be IPLEX. In further particular embodiments, thesecondary agent may be a caspase inhibitor.

In any of the embodiments described above, an effective amount of(R)-pramipexole and an effective amount of one or more of the secondaryagents described above may be provided adjunctively in separatepharmaceutical compositions or in a single dose pharmaceuticalcomposition in which the (R)-pramipexole and one or more secondary agentare combined. In such embodiments, each separate pharmaceuticalcomposition or a single dose pharmaceutical composition may furtherinclude a pharmaceutically acceptable excipient or carrier.

The compound 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole isa synthetic aminobenzothiazole derivative, having two enantiomers. The(S) enantiomer is a potent agonist of the D₂ family of dopaminereceptors, with particular affinity for the D₃ receptor subtype. As adopamine agonist, (S)-pramipexole activates dopamine receptors, thusmimicking the effects of the neurotransmitter dopamine. The(S)-pramipexole stereoisomer is a potent agonist of dopamine, with onlysmall daily doses required and indeed tolerated by patients. Bothenantiomers are thought to confer neuroprotective effects by theirability to accumulate in the brain, the spinal cord and mitochondria,and independent of the dopamine agonist activity, presumably throughinhibition of lipid peroxidation, normalization of mitochondrialfunction and/or detoxification of oxygen radicals. As such, thesecompounds may have utility as inhibitors of the cell death cascades andloss of cell viability observed in neurodegenerative diseases.

The degree to which dosing of a molecule has demonstrable phenotypicactivity resulting from affinity to particular receptors or otherpharmaco-effective proteins, even when the activity results fromaffinities to unknown targets, can be operationally defined in terms ofwhether this activity contributes in a positive way (‘on-target’activity) or a negative way (‘off-target’ activity) to a specific anddesired therapeutic effect. For any given molecule, a number of‘off-target’ activities can theoretically be identified, but ‘on-target’activity is restricted to the desired therapeutic effect. To the extentthat these activities can be measured and quantified, or comparisons bemade with known standards, an index of activity can be generated foreach of these categories (the ‘activity equivalent’, or AE), and one ormore ratios generated to compare ‘off-target’ to ‘on-target’ activities,useful to compare potential risk-benefit ratios between molecules.Without wishing to be bound by theory, the ‘off-target’ activity for(R)-pramipexole in neurodegenerative disorders (other than Parkinson'sdisease) would be the ‘on-target’ activity for its enantiomer(S)-pramipexole, used to treat PD and restless legs syndrome.

In the case of (R)-pramipexole, two activities can be defined in thiscontext. The first, which is agonist activity at a subset of humandopamine receptors and the resulting behavioral/toxicological phenotype,is ‘off-target’ activity for most neurodegenerative disorders. Thisactivity results in dose-limiting side effects due to dopamine receptoragonist activity, and for the purposes of the current discussion can bedefined to be the dopamine activity equivalent, or DAE. Throughout thisapplication, the term “dopaminergic activity equivalent” (DAE) will meanthe measure of activity at the dopamine receptors equivalent to theactivity of 1 mg of (S)-pramipexole at the dopamine receptors. Forexample, a dosage of (R)-pramipexole having a DAE of 0.01 would haveactivity at the dopamine receptors which is equivalent to the activityof 0.01 mg of (S)-pramipexole. The DAE can also be related to a varietyof pharmaceutical terms, including maximum tolerated dose (MTD), noobservable adverse effect level (NOAEL), and non-effective dose amount.For example, the NOAEL dose amount for (S)-pramipexole is mostpreferably below 0.05 mg. This, in turn, corresponds to a DAE of below0.05. A dose amount of (R)-pramipexole having a DAE of 0.01 would,therefore, be below the DAE for the most preferable (S)-pramipexoleNOAEL dose amount of 0.05 mg. In some embodiments, DAE is determined bymeasuring the binding affinity (IC₅₀) or activity (EC₅₀) at the D₂and/or D₃ receptors relative to the same parameter for 1 mg of(S)-pramipexole. For example, in certain embodiments, DAE may bedetermined by a suitable in vitro assay such as an IC₅₀ binding affinityassay for the D₂ or D₃ receptor such as those described by Schneider, C.S.; Mierau, J., “Dopamine Autoreceptor Agonists: Resolution andPharmacological Activity of 2,6-Diaminotetrahydrobenzothiazole and anAminothiazole Analogue of Apomorphine,” (1987) J. Med. Chem. 30:494-498;or Wong, S. K.-F.; Shrikhande, A. V., S. K.-F. Wong, “Activation ofExtracellular Signal-Regulated Kinase by Dopamine D2 and D3 Receptors,”(2003) Society for Neuroscience Abstracts.

Our studies suggest that the DAE for (R)-pramipexole is much lower thanmay have been previously appreciated. For example, our studies haveshown that the binding affinity for (R)-pramipexole to the D₂ and D₃dopamine receptors is about 290 and 649 times lower than(S)-pramipexole, respectively, when using high chiral purity(R)-pramipexole. By comparison, the literature reports that the bindingaffinity for the (R)-pramipexole to the D₂ dopamine receptor is about9-21 times lower than (S)-pramipexole, while the binding affinity forthe (R)-pramipexole to the D₃ dopamine receptor is about 50 times lowerthan (S)-pramipexole.

Even more striking, our studies in beagle dogs indicate that the MTDdose ratio of (R)-pramipexole to (S)-pramipexole is 10,000, while theNOAEL dose ratio of (R)-pramipexole to (S)-pramipexole is 20,000. As abiological assay, the MTD and NOAEL in dogs reveal in vivo toleranceheretofore entirely unpredictable. Because of limitations on standardand quantitative analysis, the in vivo MTD and NOAEL in dogs mayactually suggest even the slightest impurity of 0.005% could, in fact beresponsible for the dopamine agonist-related side effects. Thesecomparative studies suggest that the DAE for (R)-pramipexole is muchlower than may previously been appreciated.

The other activity of (R)-pramipexole and (S)-pramipexole isneuroprotection. Neuroprotection is a phenomenon independent ofmechanism, and hence qualifies as a category of activity. Theneuroprotective activity of (R)-pramipexole and (S)-pramipexole ismeasurable and approximately equivalent in both enantiomers. Moreover,the neuroprotective activity can be defined in relative terms as theneuroprotective activity equivalent (NAE). Neuroprotective activityequivalent (NAE) refers to the neuroprotective activity inherent in 1 mgof (S)-pramipexole. NAE can be determined, for example, by measuring theneuroprotective activity in a standard in vitro neuroprotective assayrelative to the activity of 1 mg of (S)-pramipexole. In someembodiments, the neuroprotective activity is determined by measuringcell death in the presence of MPP+ and/or rotenone in dopaminergicand/or non-dopaminergic cells (as a non-limiting example, see the assayin M. Gu, Journal of Neurochemistry, 91:1075-1081 (2004)).

Unlike the DAE, NAE has been shown to be equal in both pramipexoleenantiomers in a number of in vitro tests. However, a larger dose of apramipexole enatiomer is required to elicit neuroprotective activity invivo, and because dosages of (S)-pramipexole are limited by thedopaminergic activity of the (S) enantiomer, which can lead to adverseside effects at dosages above the “No Observable Adverse Effect Level”(NOAEL dose amount), DAE is seen as a unit measure of the potential foradverse effects when describing neuroprotection, while the NAE is seenas a unit measure of the potential for therapeutic benefit. A NOAEL doseas used herein refers to an amount of active compound or pharmaceuticalagent that produces no statistically or biologically significantincreases in the frequency or severity of adverse effects between anexposed population and its appropriate control; some effects may beproduced at this level, but they are not considered as adverse, or asprecursors to adverse effects.

In practical terms, embodiments of the invention including theadministration of (R)-pramipexole provide for significantly greater NAElevels and greater NAE/DAE levels than previously believed possible byadministration of (S)-pramipexole thereby maximizing the probabilitythat a therapeutically effective amount of (R)-pramipexole can beadministered to a patient to provide neuroprotection. The NAE and theDAE may be useful in terms of a ratio, particularly as a ratio ofbeneficial to adverse effects, and useful to define a range over which aparticular composition may be administered. (S)-pramipexole has a highDAE/NAE ratio, due to the high dopamine affinity, while thecorresponding ratio for (R)-pramipexole is significantly lower.

With respect to (S)-pramipexole, exemplary adverse events are dizziness,hallucination, nausea, hypotension, somnolence, constipation, headache,tremor, back pain, postural hypotension, hypertonia, depression,abdominal pain, anxiety, dyspepsia, flatulence, diarrhea, rash, ataxia,dry mouth, extarapyramidal syndrome, leg cramps, twitching, pharyngitis,sinusitis, sweating, rhinitis, urinary tract infection, vasodilatation,flu syndrome, increased saliva, tooth disease, dyspnea, increased cough,gait abnormalities, urinary frequency, vomiting, allergic reaction,hypertension, pruritis, hypokinesia, nervousness, dream abnormalities,chest pain, neck pain, paresthesia, tachycardia, vertigo, voicealteration, conjunctivitis, paralysis, tinnitus, lacrimation, mydriasisand diplopia. For example, a dose of 1.5 mg of (S)-pramipexole has beenshown to cause somnolence in human subjects (Public Statement onMirapex®, Sudden Onset of Sleep from the European Agency for theEvaluation of Medicinal Products; Boehringer Ingelheim product insertfor Mirapex® which indicates that the drug is administered as threedoses per day). Further, studies performed in dogs, as presented herein,(see Examples and results shown in Table 11) indicate that the NOAELdose may be as low as 0.00125 mg/kg, which is equivalent to a human doseof 0.0007 mg/kg or 0.05 mg for a 70 kg individual. Thus, with referenceto (S)-pramipexole, a NOAEL dose amount may be an amount below 1.5 mg,below 0.50 mg, or more preferably below 0.05 mg. With reference to DAEas defined herein, a NOAEL dose may have a DAE of below 1.5, below 0.5,or more preferably below 0.05.

Generally, an amount larger than the non-effective dose amount of(S)-pramipexole is necessary to have a therapeutic effect in treatingdiseases alleviated by dopamine agonist activity. This amount, however,may not be desired when a neuroprotective effect is sought, as it maylead to the described adverse side effects. A “non-effective doseamount” as used herein refers to an amount of active compound orpharmaceutical agent that elicits a biological or medicinal responsesimilar to the biological or medicinal response of a placebo as observedin a tissue, system, animal, individual or human that is being treatedby a researcher, veterinarian, medical doctor or other clinician. A“non-effective dose amount” may therefore elicit no discernabledifference from placebo in positive effects as observed in a tissue,system, animal, individual or human that is being treated by aresearcher, veterinarian, medical doctor or other clinician. As such,the “non-effective dose amount” is not expected to (1) prevent adisease; for example, preventing a disease, condition or disorder in anindividual that may be predisposed to the disease, condition or disorderbut does not yet experience or display the pathology or symptomatologyof the disease; (2) inhibit the disease; for example, inhibiting adisease, condition or disorder in an individual that is experiencing ordisplaying the pathology or symptomatology of the disease, condition ordisorder (i.e., arresting or slowing further development of thepathology and/or symptomatology), or (3) ameliorate the disease; forexample, ameliorating a disease, condition or disorder in an individualthat is experiencing or displaying the pathology or symptomatology ofthe disease, condition or disorder (i.e., reversing or reducing thepathology and/or symptomatology).

As an example, in monkeys treated with MPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), a known dopaminergicneurotoxin, (S)-pramipexole has been shown to antagonize motor deficitsand Parkinson-like symptoms in a dose-dependent manner, with the lowesteffective oral dose being 0.053 mg/kg (see Scientific Discussion athttp://www.emea.europa.eu/humandocs/PDFs/EPAR/Sifrol/059197EN6.pdf).This would be equivalent to a human dose of 0.017 mg/kg, or 1.2 mg for a70 kg individual. In human trials, the lowest effective oral dose of(S)-pramipexole with a significant effect versus placebo in thetreatment of Parkinson's disease was found to be 1.1 mg/day. Individualpatients may need doses higher than 1.1 mg/day to gain a sufficienteffect above the placebo effect (Initial Scientific Discussion for theApproval of Mirapex from the European Agency for the Evaluation ofMedicinal Products). In human trials, the lowest effective dose with asignificant effect versus placebo in the treatment of restless legssyndrome was found to be 0.25 mg/day (Boehringer Ingelheim productinsert for Mirapex®). Therefore, with reference to (S)-pramipexole, anon-effective dose amount may be an amount below 1.0 mg/day, below 0.75mg/day, below 0.5 mg/day, below 0.25 mg/day, or preferably below 0.125mg/day. With reference to DAE, a non-effective dose amount per day mayhave a DAE per day below 1.0, below 0.75, below 0.5, below 0.25, orpreferably below 0.125.

Other limits on the amount of (S)-pramipexole which can be administeredto a patient also include the maximum recommended therapeutic dose andthe maximum tolerated dose. A “maximum recommended therapeutic dose”(MRTD) refers to the dosages compiled by the FDA's Center for DrugEvaluation and Research, Office of Pharmaceutical Science, Informaticsand Computational Safety Analysis Staffs Maximum Recommended TherapeuticDose and as described in Matthews, et al., “Assessment of the HealthEffects of Chemicals in Humans: I. QSAR Estimation of the MaximumRecommended Therapeutic Dose (MRTD) and No Effect Level (NOEL) ofOrganic Chemicals Based on Clinical Trial Data,”, Current Drug DiscoveryTechnologies, 2004, 1:61-76). The FDA's MRTD database cites a MRTD forS-pramipexole of 0.1 mg/kg/day or 7.0 mg/day for a 70 lb. person.Matthews, in turn, estimates that a NOEL (no adverse effect level)usually is about one-tenth of the MRTD, which corresponds to 0.01 mg/kgor about 0.7 mg/day for a 70 lb. person.

Because of its adverse impact on naïve patients, (S)-pramipexole must betitrated over the course of weeks to reach these dosages without doselimiting adverse effects (such as that documented in BoehringerIngelheim product insert for Mirapex®). For example, for restless legsyndrome, the recommended starting daily dose amount of Mirapex® is0.125 mg taken once daily 2-3 hours before bedtime. For patientsrequiring additional symptomatic relief, the daily dose may be increasedto 0.25 mg over 4 to 7 day period and then to 0.5 mg over a second 4 to7 day period. For the treatment of Parkinson's disease, the packageinsert recommends the following titration schedule for Mirapex®:

Week Dosage (mg) Total daily dose (mg) 1 0.125 tid 0.375 2 0.25 tid 0.753 0.5 tid 1.5 4 0.75 tid 2.25 5 1.0 tid 3.0 6 1.25 tid 3.75 7 1.5 tid4.5

A “maximum tolerated dose” (MTD) as used herein refers to an amount ofactive compound or pharmaceutical agent which elicits significanttoxicity in a tissue, system, animal, individual or human that is beingtreated by a researcher, veterinarian, medical doctor or otherclinician. Single dose toxicity of (S)-pramipexole after oraladministration has been studied in rodents, dogs, monkeys and human. Inrodents, deaths occurred at doses of 70-105 mg/kg and above (InitialScientific Discussion for the Approval of Mirapex from the EuropeanAgency for the Evaluation of Medicinal Products). This is equivalent toa human dose of 7-12 mg/kg, or approximately 500-850 mg for a 70 kgindividual. In human subjects, a starting daily dose of (S)-pramipexoleof greater than 0.20 mg was not tolerated when administered to a naïvepatient. In dogs, vomiting occurred at 0.0007 mg/kg and above whilemonkeys displayed major excitation at 3.5 mg/kg. Further, the productinsert for Mirapex® sets the maximally tolerated dose for humans at 4.5mg/day, administered as three 1.5 mg single dosages. However, the 4.5mg/day dosage is not administered to a naïve patient, but, instead,reached after a titration regimen (such as that documented in theproduct insert for Mirapex®). Generally, the starting daily dosage foradministration to a naïve patient is a 0.125 mg dose administered threetimes per day and a seven-week titration schedule is recommended toreach a 1.5 mg dose administered three times daily. All species showedsigns of toxicity related to exaggerated pharmacodynamic responses to(S)-pramipexole. For example, behavioral changes including hyperactivitywere common and led to a number of secondary effects, such as reducedbody weight and other stress-induced symptoms. In minipigs and monkeys,(S)-pramipexole moderately affected cardiovascular parameters. In rats,the potent prolactin-inhibitory effect of pramipexole affectedreproductive organs (e.g. enlarged corpora lutea, pyometra), and showeda dose-related retinal degeneration during long-term exposure (InitialScientific Discussion for the Approval of Mirapex from the EuropeanAgency for the Evaluation of Medicinal Products). Studies in dogsindicate a MTD amount of (S)-pramipexole for a human subject may be anamount below 4.5 mg/day, preferably below 1.5 mg/day. Further, the MTDamount for a human subject may be an amount below 0.3 mg/dose based onresults of studies disclosed herein, and preferably below 0.2 mg/dose(see Table 11). With reference to DAE, the MTD amount may have a DAE ofbelow 1.5, below 0.3, or below 0.2.

Given the limits on the amount of (S)-pramipexole that can beadministered to a patient, the use of the embodiments of the presentinvention presents a clinically important alternative for thedevelopment of new neuroprotective therapies. The literature previouslyreported that the binding affinity of (R)-pramipexole at the D₂ receptorwas approximately 9 to 21 times less than about that of (S)-pramipexole,while the binding affinity of (R)-pramipexole at the D₃ receptor wasapproximately 50 times less than about that of (S)-pramipexole (Table10). These literature derived comparative binding affinity ratiossuggest that (R)-pramipexole can be administered only at somewhat higherdosages than (S)-pramipexole. This limitation may occur because theexquisite sensitivity of tissues, systems, animals, and human subjectsto the effects of dopamine agonism would preclude the use of(R)-pramipexole at doses that exceed tolerated doses of (S)-pramipexoleby a factor greater than the literature derived comparative bindingaffinity ratios of the two enantiomers.

The seeming preclusion of higher doses of (R)-pramipexole can bedemonstrated by reference to a theoretical 50 mg tablet. Assuming a 9times difference in binding affinities, a 50 mg tablet which is 99.95%pure would have approximately 5.575 DAE (5.55 DAE from the(R)-pramipexole and 0.025 DAE from the (S)-pramipexole). Similarly, a 25mg tablet would be expected to exhibit a DAE of 2.79 (2.78 from the(R)-pramipexole and 0.0125 DAE from the (S)-pramipexole). The MTD of(S)-pramipexole after a seven week titration regimen is 4.5 mg, or 1.5mg three times a day, which is equivalent to a 4.5 DAE in a day or 1.5DAE in a single dose. Further, the NOAEL dose amount for (S)-pramipexoleis below 1.5 mg, preferably below 0.50 mg, or more preferably below 0.05mg, which are each equivalent to 1.5 DAE, 0.5 DAE, and 0.05 DAE,respectively. Given that the single dose MTD for (S)-pramipexole has 1.5DAE and the NOAEL of (S)-pramipexole has less than about 1.5 DAE, asingle dosage of 50 mg with a DAE of 5.55 and a single dosage of 25 mgwith a DAE of 2.79 would be precluded when referring solely to theliterature derived comparative binding affinity ratios. Further, use ofa high chiral purity of 99.95% as used in these theoretical dosages,would result in unacceptably high DAEs of 5.55 and 2.79 beyond thesingle dosage MTD DAE of 1.5 mg, and far beyond the preferable NOAELs of0.5 DAE and 0.05 DAE.

To the contrary, in some embodiments, an aspect of the present inventioninvolves unexpectedly high chiral purities that have been attained.These purities have led to MTDs or NOAELs for (R)-pramipexole which arehigher than previously appreciated based on the literature derivedcomparative binding affinities. In some embodiments, pharmaceuticalcompositions, starting doses, method of treatment, and kits including(R)-pramipexole of high chiral purity are provided. Pursuant thediscussion above, a 25 mg dosage with a similar chiral purity of 99.95%would be predicted to be well above the MTD or NOAEL for (S)-pramipexoleand, therefore, result in observable adverse side effects. Studies indogs, however, suggest that the high chiral purity (R)-pramipexoleresults in NOAEL dose amounts unexpectedly than those that may have beenappreciated (Table 1). For example, a 25 mg/kg dosage of (R)-pramipexolewith no detectable amount of (S)-pramipexole (0.05% limit of detection)resulted in no observable effects in dogs, which is unexpected based onthe literature binding affinity data.

Further, studies in dogs demonstrate a high (approaching absolute)chiral purity of the pramipexole compositions for the (R)-enantiomer.(R)-pramipexole is administered in high dose levels in the studiesdisclosed herein (equivalent to human doses of 1,000 mg to 3,000 mg; seeExamples), so that even the smallest amount of (S)-pramipexole wouldcontribute to the observed NOAEL and MTD. For example, with reference tohuman equivalence doses based on data obtained in dogs, the MTD for the(R)-enantiomer has been shown to be equivalent to about 3,000 mg for a70 kg human subject, while the equivalent MTD for the (S)-enantiomerwould be equivalent to only 0.30 mg for that same subject (Table 1A).That is a difference of 10,000-fold. The NOAEL dose for the(R)-enantiomer is 20,000-fold greater than for the (S)-enantiomer (Table1A). Thus, the (R)-pramipexole compositions used in these studies mustbe at least 99.99% pure if one were to assume that the observed sideeffects stemmed only from contamination by the (S)-enantiomer. On theother hand, these data demonstrate the high dose levels of the(R)-enantiomer of pramipexole that may be administered safely. This datahighlights the usefulness of the high chiral purity for the(R)-enantiomer of pramipexole in various embodiments of the presentinvention.

Various embodiments of the invention are therefore directed tomulti-component systems, pharmaceutical compositions and methodsincluding (R)-pramipexole in higher dosages and higher chiral puritiesthan could be achieved using (S)-pramipexole without eliciting adverseeffects. Based on the chiral purity and the in vitro comparative bindingaffinity ratios, clinical NOAEL dose ratios, or clinical MTD dose ratios(herein “comparative ratios”), it may be possible to predict the DAE fora given dosage of (R)-pramipexole. Table 1 shows the DAE for a 25 mgdose of (R)-pramipexole as a function of comparative ratio and chiralpurity. These data show that a much lower DAE can unexpectedly resultfrom a 25 mg dosage form of (R)-pramipexole than may have beenpreviously appreciated, due to the lower comparative ratios describedherein when compared to the literature derived comparative ratios.

TABLE 1 DAE for a 25 mg dose of (R)-pramipexole as a function of %chiral purity and the comparative ratio Percent Chiral 20,000 10,0005,000 2,400 100 10 Purity for comparative comparative comparativecomparative comparative comparative R PPX ratio ratio ratio ratio ratioratio 99.9967 0.0020749 0.0033249 0.0058248 0.0112413 0.25081682.5007425 99.9958 0.0022999 0.0035498 0.0060498 0.0114662 0.25103952.5009450 99.9950 0.0024999 0.0037499 0.0062498 0.0116661 0.25123752.5011250 99.9933 0.0029249 0.0041783 0.0066747 0.0120909 0.25165832.5015075 99.9900 0.0037499 0.0049998 0.0074995 0.0129156 0.25247502.5022500 99.9833 0.0054248 0.0066746 0.0091742 0.0145899 0.25313332.5037575 99.9800 0.0062498 0.0074995 0.0099990 0.0154158 0.25495002.5045000 99.9750 0.0074997 0.0087494 0.0112488 0.0166641 0.25618752.5056250 99.9667 0.0095746 0.0108242 0.0133233 0.0187382 0.25824182.5074925 99.9583 0.0116745 0.0129239 0.0154229 0.0208373 0.26032082.5093825 99.9500 0.0137494 0.0149988 0.0174975 0.0229115 0.26237502.5112500 99.9333 0.0179242 0.0191733 0.0216717 0.0270847 0.26650832.5150075 99.9000 0.0262488 0.0274975 0.0299950 0.0354063 0.27475002.5225000 99.8333 0.0429229 0.0441798 0.0466666 0.0520743 0.29125832.5375075 99.8000 0.0512475 0.0524950 0.0549900 0.0603958 0.29950002.5450000 99.7500 0.0637469 0.0649938 0.0674875 0.0728906 0.31187502.5562500 99.6667 0.0845708 0.0858167 0.0883093 0.0937065 0.33249182.5749925 99.5800 0.1062448 0.1074895 0.1099790 0.1153729 0.35395002.5945000 99.5000 0.1262438 0.1274875 0.1299750 0.1353656 0.37375002.6125000 99.3333 0.1679167 0.1691583 0.1764167 0.1770222 0.41500832.6500075 99.0000 0.2512375 0.2524750 0.2549500 0.2603125 0.49750002.7250000 98.3300 0.4187291 0.4199583 0.4224165 0.4277427 0.66332502.8757500 98.0000 0.5102250 0.5024500 0.5049000 0.5102083 0.74500002.9500000 97.5000 0.62621875 0.6274375 0.629875 0.6351563 0.86875 3.0625

Table 1 attempts to illustrate the importance of both purity andaffinity on even a 25 mg single oral dosage. Assumptions regardingdopaminergic activity of the (R)-pramipexole at the dopamine receptorswould seemingly preclude even a high purity (even 100% pure) 25 mg(R)-pramipexole tablet. Based upon the disclosure of the presentinvention one can immediately envisage numerous tables to illustrate thepoint. Tables 1A and 1B below are intended to illustrate the importanceof purity for a single oral dosage form of (R)-pramipexole byillustrating the impact of even the smallest contamination of thecomposition by (S)-pramipexole

TABLE 1A “NOAEL” dosages of (R)-pramipexole compositions (based on DAE<0.05) 50 mg 100 mg 150 mg 200 mg 250 mg 500 mg (R)-purity % 99.900099.9500 99.9667 99.9750 99.9800 99.9900 (S)-impurity % 0.1000 0.05000.0333 0.0250 0.0200 0.0100 (S)-impurity DAE 0.05 0.05 0.05 0.05 0.050.05

TABLE 1B “Non-effective” dosages of (R)-pramipexole compositions (basedon DAE <0.125) 50 mg 100 mg 150 mg 200 mg 250 mg 500 mg (R)-purity %99.7500 99.8750 99.9170 99.9380 99.9500 99.9750 (S)-impurity % 0.25000.1250 0.0830 0.0620 0.0500 0.0250 (S)-impurity DAE 0.125 0.125 0.1250.125 0.125 0.125

Based on the comparative ratios for binding affinity, NOAEL and MTDvalues, it is then possible to predict the amount of (R)-pramipexolethat could be administered. Table 2 shows DAE as a function of a dosageof (R)-pramipexole (left hand column) and the comparative ratio (toprow). With reference to Table 2, a unit dose can be chosen which allowsfor an amount of (R)-pramipexole having DAE which is equal to thenon-effective amount of (S)-pramipexole. Indeed, unless a dual DAE/NAEeffect is desired, a DAE would be avoided or minimized in apharmaceutical composition. Thus, any single dose greater than 25milligrams would not be expected to avoid off-target activity and wouldbe expressly avoided by one skilled in the art. This is not true if, asin present invention, the comparative ratios exceed 200. This is bestillustrated by Table 2.

A DAE equivalent to a preferred non-effective dose amount of the(S)-pramipexole may be below 1.0 mg; more preferably below 0.5 mg, andmore preferably below 0.125 mg.

Similarly, one can ascertain the amount of (R)-pramipexole that could beadministered which would be equivalent to a no observable adverse effectlevel dose amount of the (S)-pramipexole. Table 3 shows DAE as afunction of a dosage of (R)-pramipexole (left hand column) and thecomparative ratio (top row). With reference to Table 3, a unit dose canbe chosen which allows for an amount of (R)-pramipexole having a DAEequal to the NOAEL dose amount of (S)-pramipexole. While 0.125 avoidsunwanted effects, less than 0.05 avoids NOAEL. The difference inliterature report and actual results is even more striking in Table 3.

A DAE equivalent to a preferred no observable adverse effect level(NOAEL) dose amount of the (S)-pramipexole may be below 0.5 mg,preferably below 0.05 mg.

Further, Table 4 shows DAE as a function of a dosage of (R)-pramipexole(left hand column) and the comparative ratio (top row). With referenceto Table 4, a unit dose can be chosen which allows a dose amount of(R)-pramipexole having a particular DAE.

DAE below 0.2, or below 5.

The higher comparative ratios described herein further suggest that agiven dose of (R)-pramipexole can contain a certain amount of(S)-pramipexole impurity before exceeding the acceptable DAE. Forexample, Table 3 shows that a 25 mg dose of (R)-pramipexole results in0.00125 DAE at a comparative ratio of 20,000 as suggested by the NOAELratio of (R)-pramipexole to (S)-pramipexole in the dog studies, assuminga 100% chiral purity of (R)-pramipexole. Theoretically, an additional1.4 mg of (S)-pramipexole could be added without exceeding the DAE for asingle dose MTD of (S)-pramipexole, while an additional 0.045 mg of(S)-pramipexole could be added before exceeding the preferable NOAELdose amount of (S)-pramipexole. These compositions would be 96% pure and99.8% pure. By contrast, a 25 mg of 100% pure (R)-pramipexole wouldresult in 2.78 DAE, using the comparative binding affinity ratio of 9from the literature, suggesting that even 100% purity would beinsufficient to avoid adverse side effects. Hence, the present inventionfurther provides particular doses of (R)-pramipexole which unexpectedlytolerate small amounts of (S)-pramipexole impurities.

Without wishing to be bound by theory, the neuroprotective effect of thecompositions of the present invention may derive at least in part fromthe ability of the (R)-pramipexole to prevent neural cell death by atleast one of three mechanisms: (1) the (R)-pramipexole may be reduce theformation of reactive oxygen species in cells with impairedmitochondrial energy production; (2) (R)-pramipexole may partiallyrestore the reduced mitochondrial membrane potential that has beencorrelated with Alzheimer's disease, Parkinson's disease and amyotrophiclateral sclerosis diseases; and (3) (R)-pramipexole may block the celldeath pathways which are produced by pharmacological models ofAlzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosisdiseases and mitochondrial impairment.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of embodimentsof the present invention, the preferred methods, devices, and materialsare now described.

Pharmaceutical Compositions

Various embodiments of the invention include pharmaceutical compositionsthat include an effective amount of (R)-pramipexole and an effectiveamount of one or more secondary agents. In such embodiments, theeffective amount of (R)-pramipexole and the effective amount of one ormore secondary agents may be provided adjunctively in separatepharmaceutical compositions or in a single dose pharmaceuticalcomposition. Each composition may further include a pharmaceuticallyacceptable excipient or carrier.

As such, certain embodiments of the invention provide a compositionincluding (R)-pramipexole. The compositions of the invention may include(R)-pramipexole having a chiral purity at least greater that 96%. Forexample, the chiral purity may be at least 99.5%, at least 99.6%, atleast 99.7%, at least 99.8%, at least 99.9%, least 99.95%, or preferablyat least 99.99%. In some embodiments, the composition my have a chiralpurity for (R)-pramipexole of 99.90% or greater, and in otherembodiments, the composition has a chiral purity for (R)-pramipexole of99.95% or greater. In still other embodiments, the composition has achiral purity for (R)-pramipexole of 99.99% or greater, and in certainembodiments, the chiral purity for (R)-pramipexole is 100%.

The high chiral purity of the (R)-pramipexole used herein may allow fortherapeutic compositions that have a wide individual and daily doserange. For example, in some embodiments, the amount of (R)-pramipexolemay be from about 0.01 mg/kg/day to about 10,000 mg/kg/day, from about 1mg/kg/day to about 1,000 mg/kg/day, from about 0.1 mg/kg/day to about1,000 mg/kg/day, from about 1 mg/kg/day to about 1,000 mg/kg/day, fromabout 1,000 mg/kg/day to about 10,000 mg/kg/day, or from about 1mg/kg/day to about 100 mg/kg/day. In other embodiments, the amount of(R)-pramipexole may be from about 3 mg/kg/day to about 70 mg/kg/day. Instill other embodiments, amount of (R)-pramipexole may be from about 7mg/kg/day to about 40 mg/kg/day. In yet other embodiments, the amount of(R)-pramipexole may be from about 3 mg/kg/day to about 50 mg/kg/day. Infurther embodiments, the dosage may be 10 mg/day to 1,500 mg/day, morepreferably 100 mg/day to 600 mg/day. The amount of (R)-pramipexole inthe compositions may preferably be about 25 mg to about 5,000 mg, about50 mg to about 5,000 mg, from about 100 mg to about 3,000 mg, from about300 mg to about 1,500 mg, from about 500 mg to about 1,000 mg. In yetfurther embodiments, the amount of (R)-pramipexole in the compositionsmay be about from about 25 mg to about 5,000 mg, from about 50 mg toabout 5,000 mg, from about 100 mg to about 5,000 mg, from about 200 mgto about 5,000 mg, from about 250 mg to about 5,000 mg, from about 300mg to about 5,000 mg, from about 400 mg to about 5,000 mg, from 450 mgto about 5,000 mg, from about 200 mg, to about 3,000 mg, from about 250mg to about 3,000 mg, from about 300 mg to about 3,000 mg, from about400 mg to about 3,000 mg, from 450 mg to about 3,000 mg, from about 100mg to about 1,000 mg, from about 200 mg to about 1,000 mg, from about250 mg to about 1,000 mg, from about 300 mg to about 1,000 mg, fromabout 400 mg to about 1,000 mg, from about 600 mg to about 1,000 mg, orfrom 450 mg to about 1,000 mg. In some embodiments, the amount of(R)-pramipexole is from about 600 mg to about 900 mg. This dose may beadministered as a single daily dose, or may be divided into severaldoses administered throughout the day, for example, 1 to 5 doses,preferably two or three doses per day. In some embodiments, the amountof (R)-pramipexole is from about 50 mg to about 5000 mg. In someembodiments, the amount of (R)-pramipexole is from about 100 mg to about3000 mg. In some embodiments, the amount of (R)-pramipexole is fromabout 300 mg to about 1500 mg. In some embodiments, the amount of(R)-pramipexole is from about 500 mg to about 1000 mg. In someembodiments, the composition is suitable for oral administration. Insome embodiments, the composition is a solid oral dosage form.

In some embodiments, one or more secondary therapeutic agents such as,for example, dopamine agonists, dopaminergic agonists, COMT inhibitors,MOA inhibitors, excitatory amino acid antagonists, growth factors,neurotrophic factors, antioxidants, anti-inflammatory agents,immunomodulators, anti-glutamatergics, ion channel blockers,α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptorantagonists, heat shock protein inducers/protein disaggregators anddownregulators, monoamine oxidase type B (MOAB) inhibitors, multi-targetagents, kinase inhibitors, Bcl inducers, histone deacetylase (HDAC)mediators, glial modulators, mitochondrial energy promoting agents,myostatin inhibitors, caspase inhibitors and combinations thereof may beadjunctively administered with (R)-pramipexole.

Exemplary dopamine agonists include, but are not limited to,apomorphine, carbidopa/levodopa, bromocriptine, lisuride, cabergolineand piribedel. Exemplary dopaminergic agonists include, but are notlimited to, ropinirole, rotigotine, pergolide, amantadine. ExemplaryCOMT inhibitors include, but are not limited to entacapone andtolcapone. Exemplary MOA inhibitors include, but are not limited to,selegiline, rasagiline moclobemide, isocarboxazid, phenelzine,tranylcypromine, nialamide, iproniazid, iproclozide, toloxatone,linezolid, dextroamphetamine, EVT 302 (Evotec, Inc.), Ro 19-6491(Hoffman-La Roche, Inc.), Ro 19-6327 (Hoffman-La Roche, Inc.), deprenyl,pargyline and ladostigil (TV-3326). Exemplary excitatory amino acidantagonists include, but are not limited to, talampanel.

In particular embodiments, one or more secondary agents that aredopamine agonists, such as, for example, D2/D3 agonists may beadjunctively administered with (R)-pramipexole. In such embodiments, the(R)-pramipexole exert neuroprotective effects while the D2/D3 agonistsactivate dopamine receptors. The invention is not limited to anyparticular dopamine agonist, and any dopamine agonist or combination ofdopamine agonists known in the art may be used in combination with(R)-pramipexole in embodiments of the invention. For example, usefulD2/D3 agonists may include, but are not limited to, pramipexole((6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine) (e.g.,Mirapex®), ropinirole (e.g., Requip®), carbidopa, levodopa, entacapone(e.g., COMtan®), carbidopa/levodopa (e.g., Sinemet®),carbidopa/levodopa/entacapone (e.g. Stalevo®), selegiline (e.g.,Eldpryyl®), rotigotine (e.g., Neupro®), rasagaline (e.g., Azilect®),apomorphine (e.g., Apokyn®), bromocriptine (e.g., Parlodel®), amantadine(e.g., Symmetrel®) paliroden, xaliproden, talampanel and combinationsthereof. In such embodiments, the secondary agent may have a DAE that issubstantially greater that (R)-pramipexole. Thus, the combination of(R)-pramipexole and one or more dopamine agonists may result in amulti-component system or a pharmaceutical composition that providesboth a high NAE and a high DAE and elicits both good neuroprotectiveeffects and improved dopamine activity.

For example, daily dosages of a dopamine agonist administeredadjunctively with (R)-pramipexole may a therapeutically effectiveamount, for example, from about 2 mg to about 6 mg of apomorphine, up toabout 1000 mg of levodopa, up to about 1000 mg of levodopa incombination with carbidopa, wherein the ratio of carbidopa/levodopa is1:4 or 1:10, from about 2 mg to about 24 mg of ropinorole, from about 2mg to about 6 mg of rotigotine, from about 0.05 mg to about 5 mg ofpergolide, from about 100 to about 400 mg of amantadine, from about 200mg to about 1600 mg of entacapone, from about 300 mg to about 600 mg oftolcapone, about 10 mg of selegiline, from about 0.5 mg to about 1 mg ofrasagiline.

The dopamine agonists of embodiments may provided in any suitable dosethat is therapeutically effective. For example, in some embodiments,(S)-pramipexole may be provided an amount that does not exceed about 1.0mg. In others, the non-effective dose amount of (S)-pramipexole is anamount that does not exceed about 0.75 mg, about 0.5 mg, about 0.25 mg,or about 0.125 mg. In some embodiments, the non-effective dose amount of(S)-pramipexole is less than about 0.125 mg.

In various embodiments, an effective amount of one or more growthfactors and/or neurotrophic factors such as, but are not limited to,insulin-like growth factor-1 (IGF-1), IGF-1 adenoviral-associated virus(IGF-1 AAV), mecasermin rinfabate (IPLEX), glial cell line-derivedneurotrophic factor (GDNF), hepatocyte growth factor (HGF), granulocytecolony stimulating factor (G-CSF), or combination thereof may beadjunctively administered with (R)-pramipexole. For example, someembodiments include pharmaceutical compositions that include aneffective amount of (R)-pramipexole and an effective amount of IGF-1. Insuch embodiments, the effective amount of (R)-pramipexole and theeffective amount of IGF-1 may be provided adjunctively in separatepharmaceutical compositions or in a single dose pharmaceuticalcomposition wherein each composition may further include apharmaceutically acceptable excipient or carrier.

The IGF-1 system includes three structurally related ligands (IGF-1,IGF-2 and insulin), their respective receptors, and at least six IGF-1binding proteins (IGFBP), and IGF-1 may exert multiple actions withinthe peripheral and central nervous systems. The majority of circulatingIGF-1 is bound and sequestered by IGFBPs, thereby extending thehalf-life of IGF-1, regulating its distribution, and controlling itsbioavailability in various tissues. IGF-1 functions are mediated via theIGF-1R, a hetero-tetramer having two extracellular α-subunits whichcontain the ligand binding site and two transmembrane β-subunits whichhave tyrosine kinase activity upon ligand binding and catalyze theauto-phosphorylation of tyrosine residues on the intracellular domain ofthe β-subunit. Auto-phosphorylation of the receptor causes recruitmentof the adaptor molecules insulin receptor substrate 1 and 2 (IRS1 andIRS2). Upon phosphorylation, the IRS proteins activate signalingpathways including the PI3K/Akt and p44/42 MAPK pathways.

Additional pathways linked to IGF-1 signaling include the JNK, p38 MAPK,and mTOR signaling pathways. Signaling pathways activated by IGF-1result in a wide range of cellular effects including cellularproliferation, differentiation and inhibition of apoptosis. Theneuroprotective properties of IGF-1 have been addressed in many modelsof neuronal degeneration. For example, Sakowski et al., 1 AmyotrophicLateral Sclerosis, 1-11 (2008) have shown a beneficial effects of IGF-1in neuronal cell types including human neuroblastoma cells, dorsal rootganglion cells and motor neurons. Additionally, Studies in cell linesand animal models offer insight into the potential of IGF-1 as atreatment for ALS, and in vitro studies indicate that IGF-1 may haveneuroprotective properties in ALS models. For example, in one such modelsystem, primary embryonic rat spinal motor neurons, which express IGF-IRand respond to exogenous IGF-1 treatment. Studies using primary motorneuron cultures demonstrate that IGF-1 prevents glutamate-inducedcaspase-3 cleavage, DNA fragmentation and cell death. The window for theprotective effects of IGF-1 in these studies was small followingexposure to glutamate and implies that the effects of IGF-1 might impactearly events in the activation of cell death. These studies suggest thatactivation of the PI3K/Akt and p44/42 MAPK signaling pathways may beinstrumental in the protective effects of IGF-1, as determined utilizingpathway-specific small molecule inhibitors.

The signaling pathways implicated by these studies also correlate withaxon outgrowth in postnatal corticospinal motor neurons (CSMN), whichare subject to degeneration in ALS. These studies confirm that IGF-1leads to activation of the PI3K/Akt and p44/42 MAPK signaling pathwaysin CSMN and indicate that blockade of these pathways results in defectsin axonal outgrowth. Adenoviral-associated viral (AAV)-mediatedexpression of IGF-1 in SH-SY5Y neurons and primary motor neurons alsocauses significant protection against glutamate-induced toxicity.Neighboring neurons without AAV-IGF-1 expression were also protectedagainst glutamate-induced toxicity, indicating that this delivery methodproduces biologically active IGF-1 which is released from transfectedcells. Taken together, these data indicate that IGF-1 is protective tomotor neurons, activates signaling pathways associated with cellsurvival, and promotes axonal outgrowth.

IPLEX is a hybrid protein complex of human insulin-like growth factor-1(rhIGF-1) and human insulin-like growth factor-binding protein-3(rhIGFBP-3) which is for the treatment of growth failure in childrenwith severe primary IGF-1 deficiency and which may be provide therapyfor neurodegenerative diseases such as ALS and muscular dystrophy. Glialcell line-derived neurotrophic factor (GDNF) is a small protein thatpotently promotes the survival and differentiation of dopaminergic andmotor neurons. Hepatocyte growth factor (HGF) is a paracrine cellulargrowth, motility and morphogenic factor that is secreted by mesenchymalcells and targets and acts primarily upon epithelial and endothelialcells. HGF regulates cell growth, cell motility, and morphogenesis byactivating a tyrosine kinase signaling cascade after binding to theproto-oncogene c-Met receptor and has a major role in embryonic organdevelopment, in adult organ regeneration and in wound healing. Itsability to stimulate mitogenesis, cell motility, and matrix invasion mayprovide HGF with a central role in angiogenesis and tissue regeneration.Granulocyte-colony stimulating factor (G-CSF) is a neurotrophic factorthat has been shown to protect cultured motoneurons from apoptosis thathas been shown to increase survival of motoneurons and decreasesmuscular denervation atrophy in ALS mice. G-CSF is clinically welltolerated and crosses the intact blood-brain barrier.

In various embodiments, an effective amount of one or more antioxidants,anti-inflammatories, and immunomodulators such as, but are not limitedto, AEOL 10150, cefriaxone, celastrol, coenzyme Q10, copaxone, cox-2inhibitors (including nimesulide), cyclosporin, ebselen, edaravone(radicut), promethazine, tamoxifen, thalidomide, vitamin E, VP025, orcombination thereof may be adjunctively administered with(R)-pramipexole. Without wishing to be bound by theory, substantialevidence suggests that both inflammation and oxidative damage contributeto the pathogenesis of motor neuron degeneration in ALS. Therefore,combining (R)-pramipexole with one or more antioxidants,anti-inflammatories, and immunomodulators may provide improved orsynergistic neuroprotective activity over (R)-pramipexole alone.

AEOL-10150, a small-molecule antioxidant analogous to the catalytic siteof superoxide dismutase, is a potential treatment for ALS, stroke,spinal cord injury, lung inflammation and mucositis that appears to besafe and well-tolerated in both a single and a multi-dose. Ceftriaxoneis a beta-lactam antibiotic that acts to inhibit bacterial syntheticpathways that has been shown to be a potent stimulator of glutamatereceptor (GLT1), a principal excitatory neurotransmitter in the nervoussystem whose activation is handled by the glutamate transporter (GLT1).Ceftriaxone increases expression of GLT1 in brain and up-regulatesbiochemical and functional activity of GLT-1 and appears to delay theloss of neurons and muscle strength and increase survival in the mousemodel of ALS. Glutamine is important for normal excitatory synaptictransmission, while its dysfunction is implicated in acute and chronicneurological disorders, including ALS, stroke, brain tumors andepilepsy. Celastrol is a potent anti-inflammatory compound thatsignificantly improves weight loss, motor performance and delays theonset of disease in the mouse model of ALS. Celastrol treatment.Coenzyme Q10 (CoQ10) is a mitochondrial cofactor and a powerfulantioxidant that has been shown to naturally accumulate at low levels innumber of conditions including Parkinson's disease, heart disease andcancer. It has recently been reported that CoQ10 may extend the survivalin mouse models of several neurodegenerative disorders such as ALS.

The inflammatory process in ALS appears to involve infiltration of Tcells and activation of antigen presenting cells co-localizing withmotor neuron damage in the brain and spinal cord. T cells may damagemotor neurons by cell-cell contact or cytokine secretion, or contributeindirectly to motor neuron damage through activation of microglia andmacrophages. Alternatively, T cell infiltration may be an epiphenomenonrelated to clearance of dead motor neurons. Animal models ofneuroinflammation and neurodegeneration indicate that T cell responsecan be neuroprotective or even enhance neurogenesis. Therefore, it ispossible that T cells can be induced to slow motor neuron destructionand facilitate repair in ALS. Copaxone (glatiramer acetate) is animmunomodulator that appears to induce CD8 T cell response has shownpromising results in treatment of ALS in animal models.

Cox-2 inhibitors, such as nimesulide, are a type of non-steroidalanti-inflammatory drug (NSAID) that directly target cox-2, an enzymeresponsible for inflammation and pain that have been implicated aspotential therapeutics for ALS treatment in experimental studies inanimal models, cell culture models of ALS-type neurodegeneration, andstudies of postmortem ALS brain. For example, prophylactic dietarysupplementation with nimesulide resulted in a significant delay in theonset of motor impairment and reduced cox-2-mediated induction ofpro-inflammatory prostaglandin in the cervical spinal cord toward normallevels which provides evidence for the therapeutic use of cox-2inhibitors in ALS.

Cyclosporin is an inhibitor of the mitochondrial permeability pore andan anti-anti-inflammatory/immunosuppressant that has been shown increasesurvival in animal models of ALS. Ebselen has appears to affect multiplepathways including modulation of NMDA function via the NMDA redox siteand inhibition of several kinases including p38MAPK and JNK that mayprevent binding of certain transcription factors such as AP-1. Edaravoneis a free-radical scavenger for treatment of cerebral ischemia that mayslow symptom progression and motor neuron degeneration in the ALS modelmice. Promethazine is a H1 receptor antagonist antihistamine andantiemetic that has been shown to delay the onset of ALS in mice.Tamoxifen is a selective estrogen receptor modulator used in thetreatment of breast cancer. Recently, tamoxifen has been implicated as aprotein kinase C inhibitor with anti-glutamate activity that may reducethe toxic effect of excess of glutamate on motor neurons in ALS.Thalidomide, a small molecule drug, inhibits TNF-alpha protein synthesisthat can readily cross the blood-brain barrier that may reduceinflammation. TNF-alpha plays a major role in central nervous systemneuroinflammation-mediated cell death, and thalidomide may reduce theneuroinflammation associated with TNF-alpha that is secreted by thebrain-resident marcophages (the microglial cell) in response to variousstimuli. VP025 inhibits neuro-inflammation and appear to slow theprogression of ALS through interaction with immune cells leading to themodulation of cytokines. The antioxidant vitamin E (alpha-tocopherol)has been shown to slow down the onset and progression of the paralysisin transgenic mice expressing a mutation in the superoxide dismutasegene found in certain forms of familial ALS.

In some embodiments, effective amount of one or more anti-glutamatergicsand ion channel blockers such as, but are not limited to, FP-0011,memantine, N-acetylated-a-linked acidic dipeptidease (NAALADase)inhibitors, nimodipine, riluzole or combination thereof may beadjunctively administered with (R)-pramipexole. Excessive glutamatelevels have been shown to be toxic to neurons, and evidence suggeststhat both direct and indirect glutamate toxicity may contribute to thepathogenesis of motor neuron degeneration in diseases such as ALS.

FP0011 is an antiglutamatergic compound that may reduce presynapticglutamate levels and shows strong neuroprotective properties. Memantineis a noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist thathas been shown to protect neurons against NMDA or glutamate-inducedtoxicity in vitro and in animal models of neurodegenerative diseases.N-Acetylated-Alpha-Linked-Acidic-Dipeptidase (NAALADase) convertsN-Acetyl-Aspartyl-Glutamate into glutamate during neuronal damage andmay represent a new approach to block the release of excess glutamatewithout interfering with normal brain function in treatment ofneurodegenerative disorders. Nimodipine is a dihydropyridine calciumchannel blocker which may antagonize excitatory amino acid receptoractivation decreasing calcium entry into damaged neurons and might helpto slow or reverse ALS. Riluzole is an anti-convulsant and aneuroprotective agent that specifically blocks sodium channels in theirinactivated states. Riluzole has been shown to significantly prolongsurvival in the bulbar-onset group of the overall population.

In other embodiments, an effective amount of one or more AMPA receptorantagonists such as, but are not limited to,1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide(NBQX), talampanel, or combination thereof may be adjunctivelyadministered with (R)-pramipexole. AMPA antagonists have shown clearbeneficial effect in mouse models of ALS, including prolonged survivaland maintained or improved motor function.

1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide(NBQX) is an AMPA receptor antagonist that may reduce excitotoxicitywhich has been implicated in the selective motor neuron loss in ALS.Talampanel is a selective AMPA receptor antagonist that preventsglutamate excitotoxicity which may trigger motor neuron death.Talampanel has been studied in a small group of human subjects with ALS,showing a positive trend in the ALS Functional Rating Scale as comparedto those treated with placebo.

In still other embodiments, an effective amount of one or more heatshock protein inducers, protein disaggregators or protein downregulatorssuch as, but are not limited to, arimoclomol, ISIS 333611, lithium,misfolded SOD-1 antibodies, rhHSP70, TDP-43 antagonists and trehalose orcombinations thereof may be adjunctively administered with(R)-pramipexole.

Arimoclomol has been shown to protect motor nerves subjected to physicaltrauma and accelerate the regeneration of previously damaged nerves inanimals by amplifying “molecular chaperone” proteins thereby enhancing acell's natural ability to mend damaged, misfolded proteins. Thus,Arimoclomol may provide cellular protection from misfolded, toxicproteins that are believed to cause many neurodegenerative diseases andmay have broad application profile in various neurodegenerative diseasesincluding, for example, ALS. ISIS 333611 inhibits Cu/Zn superoxidedismutase (SOD1) a molecule associated with the familial form of ALS,and delivery of ISIS 333611 directly to the cerebral spinal fluidsignificantly decreases production of the mutant protein in neurons andsurrounding cells and has been shown to prolong the lifetime of ratsthat show features of ALS. Lithium has been shown to possessneuroprotective properties in a variety of disease models such as brainischemia and kainite toxicity and appears to have the ability to promoteautophagy through the inhibition of inositol-monophosphatase 1. Indisease models of ALS, lithium delays disease onset and augments lifespan through the activation of autophagy, increasing in the number ofmitochondria in motor neurons and suppressing reactive astrogliosis.

Misfolding of SOD1 has emerged as mechanism underlying motor neurondegeneration in individuals with ALS who carry a mutant SOD1 gene. TheSOD1 exposed dimer interface (SEDI) antibody recognizes only those SOD1conformations in which the native dimer is disrupted or misfolded, andby binding specifically to the misfolded protein, SEDI has been shown toameliorate mutant protein deposition in the cytoplasm and augment lifespan.

One of the most common cellular mechanisms to survive a stressfulcondition is the heat shock response, characterized by an increase inthe transcription of a subset of genes resulting in the production ofinducible heat shock proteins, and insufficient availability of HSP70may contribute to motorneuron death in ALS mice. HSP70 expression hasbeen shown to confer neuroprotection, and exogenous delivery ofrecombinant human HSP70 (rhHSP70) has been shown to increase life span,delay symptom onset, preserve motor function, and prolong motorneuronsurvival in mouse models of ALS.

Evidence suggests a pathophysiological link between ubiquitinated TARDNA binding protein (TDP-43) and ALS, and TDP-43 inclusions have beenshown to play a role in the pathogenesis of ALS. Therefore, TDP-43antagonists may have a therapeutic effect on neurodegenerative diseases.Trehalose is a natural alpha-linked disaccharide formed by an α, α-1,1-glucoside bond between two α-glucose units that may inhibitpolyglutamine-induced protein aggregation in vitro and in vivo inHuntington's disease models.

In yet other embodiments, an effective amount of one or more MOABinhibitor such as, but are not limited to, R(+)N-propargyl-1-aminoindan(rasagiline) may be adjunctively administered with (R)-pramipexole.Rasagiline is an irreversible inhibitor of MOAB that has been shown tohave a significant dose-dependent therapeutic effect on both preclinicaland clinical motor function and survival in neurodegenerative disorders.

In further embodiments, with an effective amount of one or moremulti-target agents such as, but are not limited to,4-[2(aminomethyl)-1,3-thiazol-4-yl]-2,6 di-tert-butylphenol (BN82451)may be adjunctively administered with (R)-pramipexole. BN82451 belongsto a family of small molecules designated as multi-targeting or hybridmolecules that is orally active, penetrates central nervous system, andelicits potent neuronal protection, due to Na+ channel blockade,antioxidant properties, and mitochondria-protecting activity, andanti-inflammatory properties due to inhibition of cyclooxygenases.

In still further embodiments, an effective amount of one or more kinaseinhibitors such as, but are not limited to, olomoucine,quinolin-2(1H)-one derivatives, roscovitine, tamoxifen and combinationsthereof may be adjunctively administered with (R)-pramipexole.Misfolding of SOD1 leads to protein aggregation which has been observedin animal models of ALS as well as in human sporadic and familial ALSpatients and is one of the earliest measurable events in the mouse. Theprotein aggregates may also impair or alter the function of theproteasome, the cell's protein degradation machinery, which, in turn,may alter the half-life of key cell cycle regulatory proteins including,for example, cyclin dependent kinases (CDKs) which control the cell'sdivision cycle. In fact, post mortem examinations of human ALS patientsindicate that of CDK4 and CDK6 concentrations may be increased inneurons and CDK2 concentrations may be increased in astrocytes.Furthermore, activation and proliferation of astrocytes and microglia inhumans and the mouse model astrocytes and microglia raise thepossibility that generalized, non-specific inflammation and release ofproliferative factors can directly cause aberrant cell cycle reentry andapoptosis in neurons as a result of increased CDK2 concentrations.Therefore, inhibition of cell cycle re-entry by inhibiting kinases suchas, for example, CDK2, CDK4, and CDK6 could protect neurons fromapoptosis, and might protect against astrocyte and microglialproliferation. Olomoucine is a known inhibitor of CDK2 and CDK5 that hasbeen shown to arrests cells both in late G1 and at the G2/M transition(prophase/metaphase) of the cell cycle. Quinolin-2(1H)-one derivativesselectively inhibit cyclin-dependent kinase 5 (CDK5) which has beenimplicated in a number of neurodegenerative disorders such as, forexample, Alzheimer's disease, ALS, and ischemic stroke. Roscovitine is apotent inhibitor of CDK1, CDK2, and CDK5 that has been shown to mediatesneuropathology in Nieman's Pick Type C disease (a fatalneurodegenerative disorder). Tamoxifen has been widely used as aselective estrogen receptor modulator in the treatment of breast cancer,and a new mode of action for tamoxifen has been discovered whichimplicates it as a protein kinase C inhibitor with anti-glutamateactivity. Excess of glutamate is believed to play a role in ALS due toits toxic effect on motor neurons.

In yet further embodiments, an effective amount of one or more agentsBcl inducers such, but are not limited to, ginsenoside Rb1, ginsenosideRg1, G3139, oblimersen, and combinations thereof may be adjunctivelyadministered with (R)-pramipexole. Ginsenosides Rb1 and ginsenoside Rg1are extracted from ginseng root and appear to protect spinal neuronsfrom excitotoxicity induced by glutamate and kainic acid, as well asoxidative stress, by stimulating Bcl expression. G3139 (oblimersen) isan 18-mer phosphorothioate oligo deoxy ribonucleotide antisensemolecule, which is targeted to the initiation codon region of the Bcl-2mRNA and effectively inhibits Bcl-2 transcription. Bcl-2 is apredominately integral membrane protein that is found in the outermitochondrial membrane, endoplasmic reticulum, or outer nuclear membranethat is capable of forming ion channels in artificial membranes and canblock the release of cytochrome c into the cytosol at least in part bycorrecting a defect in ATP/ADP exchange across the mitochondrialmembrane. Cytochrome c forms an “apoptosome” complex with ATP, Apaf-1,and pro-caspase 9, which leads to the cleavage of the latter into anactive peptide.

In some embodiments, an effective amount of one or more histonedeacetylase (HDAC) inhibitors such as, but are not limited to,phenylbutyrate, scriptaid, valproic acid, and combinations thereof maybe adjunctively administered with (R)-pramipexole. Transcriptiondysregulation may play a role in the pathogenesis of ALS. HDACinhibitors can “desilence” a genome, allowing expression of genes thatunder normal situations would not be expressed thereby reversing thisdysregulation. Sodium phenylbutyrate is an HDAC inhibitor that improvestranscription and post-transcriptional pathways and promotes cellsurvival in a mouse model of motor neuron disease. Scriptaid is an HDACinhibitor that changes the expression profile of protein factors thatare involved in the recognition and binding of protein aggregates by thedynactin complex. Sodium valproate is an HDAC inhibitor that has beendemonstrated to inhibit microglial cells hyperactivity and production ofinflammatory mediators, appears to have protective effects in thesuperoxide dismutase (SOD) in mouse model of ALS, and stimulates stemcell activity, which may allow for regeneration of damaged motorneurons.

In other embodiments, an effective amount of one or more glialmodulators such as, but are not limited to, ONO-2506 may be adjunctivelyadministered with (R)-pramipexole. ONO-2506 lowers the potential forglutamate excitotoxicity by increasing the activity of glutamatetransporters in astrocytes and has been shown to inhibit expansion ofcerebral infarction by modulating the function of astrocytes. Earlystudies suggest that ONO-2506 may exhibit a neuroprotective effect bypreventing irreversible injury to neurons in the brain.

In yet other embodiments, an effective amount of one or moremitochondrial energy promoters such as, but are not limited to,resveratrol, creatine, erythropoietin, cholest-4-en-3-One, oxime(TRO-19622) and combinations thereof may be adjunctively administeredwith (R)-pramipexole. Resveratrol is a powerful antioxidant found in redgrape skins that has been found to suppress the influx of calcium intocells associated with glutamate-induced cell toxicity. Creatine aids inthe formation of ATP, the primary source of cellular energy in the body,and has been shown to provide protective mechanisms againstneurodegenerative disorders by stabilizing cellular membranes andmitochondrial energy-transfer complexes which may reduce motor neurondeath by improving mitochondrial function. Creatine may also reduceoxidative stress and increase glutamate uptake and may help reduce theloss of muscle strength in ALS patients. Erythropoietin (EPO) is aglycoprotein hormone that controls erythropoiesis, red blood cellproduction that has recently been identified as a cytokine with variousneuroprotective effects, including, for example, reduction ofinflammation, enhancement of survival signals, and prevention ofneuronal cell death. Cholest-4-en-3-one, oxime (TRO-19622) is a lowmolecular-weight compound shown to enhance motor neuron survival andgrowth by interacting with protein components of the mitochondrialpermeability transition pore and that may rescue motor neuron cellbodies from axonomy-induced cell death in vivo.

In still other embodiments, an effective amount of one or more myostatininhibitors such as, but are not limited to, ACE-031, MYO-029 andcombinations thereof may be adjunctively administered with(R)-pramipexole. Myostatin is a negative regulator of muscle mass andinhibiting myostatin the body may be free to rebuild muscle tissue. Thefunctional improvement of dystrophic muscle by myostatin blockade mayprovide a novel pharmacological strategy for treatment of diseasesassociated with muscle wasting such as DMD and ALS. ACE-031 is amyostatin inhibitor that was developed to treat diseases involving theloss of muscle mass, strength and function in diseases includingmuscular dystrophy, ALS, and cancer-related muscle loss. MYO-029 is arecombinant human antibody designed to bind to and inhibit the activityof myostatin.

In certain embodiments, an effective amount of one or more caspaseinhibitors such as, but are not limited to, ESPA-1002, IDN-6556,pralnacasan, and combinations thereof may be adjunctively administeredwith (R)-pramipexole. Caspases are the mammalian cell death effectorproteins that appear to be up-regulated in ALS. Apoptosis, programmedcell death, has been demonstrated to occur in the CNS, following bothacute injury and during chronic neurodegenerative conditions such asALS, and caspase inhibition has been demonstrated to be therapeuticallyeffective in moderating excessive apoptosis. Therefore, targetedinhibition of caspases may represent a potential therapeutic option fortreatment of neurodegenerative disorders. ESPA-1002 is a specificinhibitor of caspase 8 and caspase 9. IDN-6556 is a caspase inhibitorthat has been shown to slow progression of ALS. Pralnacasan is aninhibitor of the inflammatory caspase interleukin-1-converting enzyme(ICE) and has been shown to significantly slow symptom progression ofALS.

Various embodiments of the invention are directed to multi-componenttherapeutics, multi-component systems, and pharmaceutical compositionsin which (R)-pramipexole and one or more secondary therapeutic agentssuch as, for example, those listed above are provided in separate unitdoses that are administered to a patient in need of treatment inseparate, individual pharmaceutical compositions. Various otherembodiments are directed to multi-component therapeutics,multi-component systems, and pharmaceutical compositions the include(R)-pramipexole and one or more secondary therapeutic agents such asthose listed above in a single unit dose form that can be administeredto a patient in need of treatment in a single pharmaceuticalcomposition. In such embodiments, the separate, individualpharmaceutical compositions and the single pharmaceutical compositionincluding both (R)-pramipexole and one or more secondary therapeuticagents may include one or more pharmaceutically acceptable adjuvant,carrier or excipient.

The multi-component therapeutics, multi-component systems, andpharmaceutical compositions are not limited by the type or secondarytherapeutic agent and any of the secondary agents described above may beuseful in embodiments of the invention. However, in particularembodiments, the secondary agent may be a dopamine agonist. For example,in one exemplary embodiment, the dopamine agonist may be ropinirole(Requip®), and in another exemplary embodiment, the dopamine agonist maybe carbidopa/levodopa (Sinemet®). In other particular embodiments, thesecondary agent may be an anti-glutamatergic. For example, in oneexemplary embodiment, the secondary agent may be riluzole (Rilutek®). Inyet other particular embodiments, the secondary agent may be anexcitatory amino acid. For example, in one exemplary embodiment, thesecondary agent may be talampanel. In still other particularembodiments, the secondary agent may be a growth factor. For example, inone exemplary embodiment, the secondary agent may be IPLEX. In furtherparticular embodiments, the secondary agent may be a caspase inhibitor.

The multi-component therapeutics, multi-component systems, andpharmaceutical compositions, of some embodiments, may generally be usedas neuroprotectants or to provide neuroprotection in a patient to whichthe compositions are administered. In other embodiments, themulti-component therapeutics, multi-component systems, andpharmaceutical compositions may be useful in the treatment of diseasesrelated to neuronal degeneration or neuronal cell death or to alleviatethe symptoms of such diseases by the action of a neuroprotectant. Instill other embodiments, the multi-component therapeutics,multi-component systems, and pharmaceutical compositions of theinvention may additionally be useful for restoring or improvingneuronal, retinal and muscle function in patients, to treatneurodegenerative diseases, or diseases associated with mitochondrialdysfunction or increased oxidative stress, and in particularembodiments, the multi-component therapeutics, multi-component systems,and pharmaceutical compositions may be used to treat neurodegenerativedementias, neurodegenerative movement disorders, ataxia, seizuredisorders, motor neuron disorders or diseases, and inflammatorydemyelinating disorders in patients.

Embodiments of the invention are not limited to a particular mode ofaction. For instance, in some embodiments, the multi-componenttherapeutics, multi-component systems, and pharmaceutical compositionsof the invention may be effective as inhibitors of oxidative stress orlipid peroxidation and may be useful in detoxification of oxygenradicals and normalization of mitochondrial function which may be usefulin the treatment of numerous diseases. For example, increases inreactive oxygen species and other free radicals have been associatedwith ALS and at least in some cases may be the result of mutations inthe SOD-1 gene which destroys superoxide radicals. In fact, the SOD-1enzyme may play a pivotal role in the pathogenesis and progression offamilial amyotrophic lateral sclerosis (FALS) which make up about 10% ofall ALS patients are familial cases and of which 20% have mutations inthe SOD-1 gene. Moreover, recent studies have linked premature neuronaldeath associated with ALS to mutated mitochondrial genes which lead toabnormalities in functioning of the energy production pathways inmitochondria. In other embodiments, the multi-component therapeutics,multi-component systems, and pharmaceutical compositions may beeffective as treatment for impaired motor function in, for example,cardiac and striated muscle and retinal tissues which is associated withvarious degenerative diseases and neurodegenerative diseases such asALS, Parkinson's disease, Alzheimer's disease, and macular degeneration.For example, in certain embodiments, the multi-component therapeutics,multi-component systems, and pharmaceutical compositions of theinvention may be used in the treatment of age related maculardegeneration. In such embodiment, the multi-component therapeutics,multi-component systems, and pharmaceutical compositions suitable forsystemic administration, ocular administration or topical administrationto the eye may be prepared. Therefore, the multi-component therapeutics,multi-component systems, and pharmaceutical compositions including(R)-pramipexole and the secondary agents provided above may be wellsuited for treatment of ALS, Parkinson's Disease, Alzheimer's disease,and macular degeneration. Any listing of disorders or diseases providedin discussion of the invention embodied herein is for exemplary purposesonly and is not limiting in any way. Therefore, the compositions of theinvention may be useful in the treatment of numerous unlisted disorders.

Further embodiments of the invention are directed methods ofameliorating the symptoms associated with degenerative andneurodegenerative diseases such as, for example, ALS, Parkinson'sDisease, Alzheimer's disease, and macular degeneration by administering(R)-pramipexole and one or more secondary therapeutic agents such asdopamine agonists, dopaminergic agonists, COMT inhibitors, MOAinhibitors, excitatory amino acid antagonists, growth factors,neurotrophic factors, antioxidants, anti-inflammatory agents,immunomodulators, anti-glutamatergics, ion channel blockers,α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptorantagonists, heat shock protein inducers/protein disaggregators anddownregulators, monoamine oxidase type B (MOAB) inhibitors, multi-targetagents, kinase inhibitors, Bcl protein inducers, histone deacetylase(HDAC) mediators, glial modulators, mitochondrial energy promotingagents, myostatin inhibitors, caspase inhibitors and combinationsthereof. For example, in some embodiments, the rate of cell deathassociated with neurodegenerative disorders such as Parkinson's diseaseand/or ALS may be reduced by administering the multi-componenttherapeutics, multi-component systems, and pharmaceutical compositionsof various embodiments described above.

The pharmaceutical compositions of various embodiments may be suitablefor oral administration, and in some embodiments, may be in a solid oraldosage form such as, for example, a tablet or a capsule.

In particular embodiments, the multi-component therapeutics,multi-component systems, and pharmaceutical compositions of theinvention may include at least about 25 mg of (R)-pramipexole and lessthan about about 1.5 dopaminergic activity equivalents (“DAE”). Table 1shows the DAE for a 25 mg dose of (R)-pramipexole as a function of aparticular chiral purity of the (R)-pramipexole in the dose and thecomparative binding affinity ratio. The embodiments for the amount of(R)-pramipexole in the multi-component therapeutics, multi-componentsystems, and pharmaceutical compositions, the DAE, chiral purity, anddosage form, which are described herein separately for the sake ofbrevity, can be joined in any suitable combination.

The chiral purity of the (R)-pramipexole in the multi-componenttherapeutics, multi-component systems, and pharmaceutical compositionsof various embodiments of the invention may be at least 99.5%, at least99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.95%,or in particular embodiments, at least 99.99%. In certain embodiments,the chiral purity for (R)-pramipexole may be 100% or as close to 100% ascan be measured. In some embodiments, the composition has a chiralpurity for (R)-pramipexole of 99.9% or greater. In other embodiments,the composition has a chiral purity for (R)-pramipexole of 99.95% orgreater. In still other embodiments, the composition has a chiral purityfor (R)-pramipexole of 99.99% or greater.

In some embodiments, the multi-component therapeutics, multi-componentsystems, and pharmaceutical compositions of the invention may includeless than about about 0.5 dopaminergic activity equivalents (DAE). Inother embodiments, the multi-component therapeutics, multi-componentsystems, and the multi-component therapeutics, multi-component systems,and pharmaceutical compositions of the invention of the invention mayinclude less than about 0.05 DAE. These DAE values are derived from theno observable adverse effect levels of (R)-pramipexole as discussedabove. In some embodiments, the multi-component therapeutics,multi-component systems, and pharmaceutical compositions of theinvention has a DAE which is less than the DAE as calculated from themaximum tolerated dose (MTD) amount or non-effective dose amounts of(S)-pramipexole. With reference to non-effective dose amounts of(S)-pramipexole, in some embodiments, the DAE does not exceed about 1.0,does not exceed about 0.75, does not exceed about 0.5, does not exceedabout 0.25, or does not exceed about 0.125. With reference to MTDamount, the composition may have a DAE of below 1.5, below 0.3, or below0.2.

In some embodiments, the multi-component therapeutics, multi-componentsystems, and pharmaceutical compositions may include at least about 50mg of (R)-pramipexole. In other embodiments, the multi-componenttherapeutics, multi-component systems, and pharmaceutical compositionsmay include at least about 75 mg of (R)-pramipexole. In still otherembodiments, multi-component therapeutics, multi-component systems, andpharmaceutical compositions may include at least about 125 mg of(R)-pramipexole. In yet other embodiments, the multi-componenttherapeutics, multi-component systems, and pharmaceutical compositionsmay include at least about 150 mg of (R)-pramipexole. In furtherembodiments, the multi-component therapeutics, multi-component systems,and pharmaceutical compositions may include at least about 200 mg of(R)-pramipexole. In yet further embodiments, the multi-componenttherapeutics, multi-component systems, and pharmaceutical compositionsmay include at least about 250 mg of (R)-pramipexole. In still furtherembodiments, the multi-component therapeutics, multi-component systems,and pharmaceutical compositions may include at least about 300 mg of(R)-pramipexole. In some embodiments, the multi-component therapeutics,multi-component systems, and pharmaceutical compositions may include atleast about 400 mg of (R)-pramipexole. In other embodiments, themulti-component therapeutics, multi-component systems, andpharmaceutical compositions may include at least about 500 mg of(R)-pramipexole. In still other embodiments, the multi-componenttherapeutics, multi-component systems, and pharmaceutical compositionsmay include at least about 600 mg of (R)-pramipexole. In yet otherembodiments, the multi-component therapeutics, multi-component systems,and pharmaceutical compositions may include at least about 750 mg of(R)-pramipexole. In some embodiments, the multi-component therapeutics,multi-component systems, and pharmaceutical compositions may include atleast about 1000 mg of (R)-pramipexole.

In certain embodiments, the amount of (R)-pramipexole (mg) administeredper kg body weight of the patient per day through the course oftreatment using the multi-component therapeutics, multi-componentsystems, and pharmaceutical compositions of the invention may be fromabout 0.01 mg/kg/day to about 10,000 mg/kg/day, from about 1 mg/kg/dayto about 1,000 mg/kg/day, from about 0.1 mg/kg/day to about 1,000mg/kg/day, from about 1 mg/kg/day to about 1,000 mg/kg/day, from about1,000 mg/kg/day to about 10,000 mg/kg/day, or from about 1 mg/kg/day toabout 100 mg/kg/day. In some embodiments, the amount of (R)-pramipexolemay be from about 3 mg/kg/day to about 70 mg/kg/day. In someembodiments, the amount of (R)-pramipexole may be from about 7 mg/kg/dayto about 40 mg/kg/day. In some embodiment, the amount of (R)-pramipexolemay be from about 3 mg/kg/day to about 50 mg/kg/day. In someembodiments, the dosage may be 10 mg/day to 1,500 mg/day, morepreferably 100 mg/day to 600 mg/day. The amount of (R)-pramipexole inthe compositions may be about 50 mg to about 5,000 mg, from about 100 mgto about 3,000 mg, from about 300 mg to about 1,500 mg, from about 500mg to about 1,000 mg. In some embodiments, the amount of (R)-pramipexolein the compositions may be about from about 25 mg to about 5,000 mg,from about 50 mg to about 5,000 mg, from about 100 mg to about 5,000 mg,from about 200 mg to about 5,000 mg, from about 250 mg to about 5,000mg, from about 300 mg to about 5,000 mg, from about 400 mg to about5,000 mg, from 450 mg to about 5,000 mg, from about 200 mg, to about3,000 mg, from about 250 mg to about 3,000 mg, from about 300 mg toabout 3,000 mg, from about 400 mg to about 3,000 mg, from 450 mg toabout 3,000 mg, from about 100 mg to about 1,000 mg, from about 200 mgto about 1,000 mg, from about 250 mg to about 1,000 mg, from about 300mg to about 1,000 mg, from about 400 mg to about 1,000 mg, from about600 mg to about 1,000 mg, or from 450 mg to about 1,000 mg. In someembodiments, the amount of (R)-pramipexole is from about 600 mg to about900 mg. In some embodiments, the amount of (R)-pramipexole is from about50 mg to about 5000 mg. In some embodiments, the amount of(R)-pramipexole is from about 100 mg to about 3000 mg. In someembodiments, the amount of (R)-pramipexole is from about 300 mg to about1500 mg. In some embodiments, the amount of (R)-pramipexole is fromabout 500 mg to about 1000 mg.

In some embodiments, a starting daily dose of (R)-pramipexole may beequal to the daily dose of (R)-pramipexole administered throughout thecourse of treatment. For example, in embodiments in which the daily doseof (R)-pramipexole is 5000 mg/day, the starting daily dose may be 5000mg per day, and this daily dose may be maintained throughout treatment.In other embodiments, (R)-pramipexole may be titrated such that thestarting daily dose may of (R)-pramipexole may be less than 5000 mg, forexample, 500 mg or 100 mg, and the daily dose may be increased everyday, every other day, or per week until the required daily dose of, forexample, 5000 mg is reached. In such embodiments, the starting dailydose may vary. For example, in some embodiments, the starting daily dosemay be at least about 25 mg of (R)-pramipexole. In some embodiments, thestarting daily dose may be at least about 50 mg of (R)-pramipexole. Insome embodiments, the starting daily dose may be at least about 75 mg of(R)-pramipexole. In some embodiments, the starting daily dose may be atleast about 125 mg of (R)-pramipexole. In some embodiments, the startingdaily dose may be at least about 150 mg of (R)-pramipexole. In someembodiments, the starting daily dose may be at least about 200 mg of(R)-pramipexole. In some embodiments, the starting daily dose may be atleast about 300 mg of (R)-pramipexole. In some embodiments, the startingdaily dose may be at least about 400 mg of (R)-pramipexole. In someembodiments, the starting daily dose may be at least about 500 mg of(R)-pramipexole. In some embodiments, the starting daily dose may be atleast about 600 mg of (R)-pramipexole. In some embodiments, the startingdaily dose may be at least about 750 mg of (R)-pramipexole. In someembodiments, the starting daily dose may be at least about 1000 mg of(R)-pramipexole. In some embodiments, the starting daily dose may befrom about 600 mg to about 900 mg of (R)-pramipexole. In still otherembodiments, the a starting daily dose of (R)-pramipexole may be greaterthan the daily dose of (R)-pramipexole administered throughout thecourse of treatment. For example, in some embodiments the starting dailydose may be about 5000 mg and the daily dose may be decreased every day,every other day, or per week until the required daily dose of, forexample, 500 or 100 mg is reached.

The starting daily dose amount of (R)-pramipexole in the compositionsmay preferably be about 50 mg to about 5,000 mg, from about 100 mg toabout 3,000 mg, from about 300 mg to about 1,500 mg, from about 500 mgto about 1,000 mg. In some embodiments, the starting daily dose amountof (R)-pramipexole in the compositions may be about from about 25 mg toabout 5,000 mg, from about 50 mg to about 5,000 mg, from about 100 mg toabout 5,000 mg, from about 200 mg to about 5,000 mg, from about 250 mgto about 5,000 mg, from about 300 mg to about 5,000 mg, from about 400mg to about 5,000 mg, from 450 mg to about 5,000 mg, from about 200 mg,to about 3,000 mg, from about 250 mg to about 3,000 mg, from about 300mg to about 3,000 mg, from about 400 mg to about 3,000 mg, from 450 mgto about 3,000 mg, from about 100 mg to about 1,000 mg, from about 200mg to about 1,000 mg, from about 250 mg to about 1,000 mg, from about300 mg to about 1,000 mg, from about 400 mg to about 1,000 mg, fromabout 600 mg to about 1,000 mg, or from 450 mg to about 1,000 mg. Insome embodiments, the starting daily dose amount of (R)-pramipexole isfrom about 600 mg to about 900 mg. This dose may be administered as asingle daily dose, or may be divided into several doses administeredthroughout the day, for example, 1 to 5 doses per day, preferably two tothree doses per day. In some embodiments, the starting daily dose amountof (R)-pramipexole is from about 50 mg to about 5000 mg. In someembodiments, the starting daily dose amount of (R)-pramipexole is fromabout 100 mg to about 3000 mg. In some embodiments, the starting dailydose amount of (R)-pramipexole is from about 300 mg to about 1500 mg. Insome embodiments, the starting daily dose amount of (R)-pramipexole isfrom about 500 mg to about 1000 mg.

In such embodiments, the starting daily dose amount of (R)-pramipexolemay be from about 0.01 mg/kg/day to about 10,000 mg/kg/day, from about 1mg/kg/day to about 1,000 mg/kg/day, from about 0.1 mg/kg/day to about1,000 mg/kg/day, from about 1 mg/kg/day to about 1,000 mg/kg/day, fromabout 1,000 mg/kg/day to about 10,000 mg/kg/day, or from about 1mg/kg/day to about 100 mg/kg/day. In some embodiments, the startingdaily dose amount of (R)-pramipexole may be from about 3 mg/kg/day toabout 70 mg/kg/day. In some embodiments, the starting daily dose amountof (R)-pramipexole may be from about 7 mg/kg/day to about 40 mg/kg/day.In some embodiment, the starting daily dose amount of (R)-pramipexolemay be from about 3 mg/kg/day to about 50 mg/kg/day. In someembodiments, the starting daily dose amount may be 10 mg/day to 1,500mg/day, more preferably 100 mg/day to 600 mg/day.

The amount of each, individual secondary agent of the one or moresecondary agents in the pharmaceutical compositions of the invention mayvary depending on, for example, the secondary agent utilized. In someembodiments, the amount of the one or more secondary agent in thepharmaceutical composition may be the amount suggested by themanufacture as a daily dose, starting daily dose, or dose per kg patientweight, per day. For example, the amount of each, individual secondaryagent of the one or more secondary agents in the compositions of variousembodiments of the invention may be from about 2 mg to about 5,000 mg,from about 10 mg to about 3,000 mg, from about 30 mg to about 1,500 mg,from about 50 mg to about 1,000 mg. In some embodiments, the amount ofeach, individual secondary agent of the one or more secondary agents insuch compositions may be about from about 25 mg to about 5,000 mg, fromabout 50 mg to about 5,000 mg, from about 100 mg to about 5,000 mg, fromabout 200 mg to about 5,000 mg, from about 250 mg to about 5,000 mg,from about 300 mg to about 5,000 mg, from about 400 mg to about 5,000mg, from 450 mg to about 5,000 mg, from about 200 mg, to about 3,000 mg,from about 250 mg to about 3,000 mg, from about 300 mg to about 3,000mg, from about 400 mg to about 3,000 mg, from 450 mg to about 3,000 mg,from about 100 mg to about 1,000 mg, from about 200 mg to about 1,000mg, from about 250 mg to about 1,000 mg, from about 300 mg to about1,000 mg, from about 400 mg to about 1,000 mg, from about 600 mg toabout 1,000 mg, or from 450 mg to about 1,000 mg. In some embodiments,the amount of each, individual secondary agent of the one or moresecondary agents may be from about 600 mg to about 900 mg. In someembodiments, the amount of each, individual secondary agent of the oneor more secondary agents may be from about 50 mg to about 5000 mg. Insome embodiments, the amount of each, individual secondary agent of theone or more secondary agents may be from about 100 mg to about 3000 mg.In some embodiments, the amount of each, individual secondary agent ofthe one or more secondary agents may be from about 300 mg to about 1500mg. In some embodiments, the amount of each, individual secondary agentof the one or more secondary agents may be from about 500 mg to about1000 mg.

In some embodiments, the multi-component therapeutics, multi-componentsystems, and pharmaceutical compositions of various embodiments of theinvention may include, for example, microcrystalline cellulose,mannitol, croscarmellose sodium, magnesium stearate, or combinationthereof. For example, certain embodiments of the invention include apharmaceutical formulation including (R)-pramipexole and, in someembodiments, one or more secondary agents, about 20% to about 50% byweight of the formulation of one or more microcrystalline cellulose;about 10% to about 30% by weight mannitol; about 2% to about 6%crospovidone; and about 0.01% to about 2% magnesium stearate. In someembodiments, the pharmaceutical composition may include a diluent in anamount from about 20% to about 50% by weight of the formulation, and incertain embodiments, the formulation may include about 10% to about 30%by weight of a second diluent. In still other embodiments, theformulation may include about 2% to about 6% of a disintegrant and inyet other embodiments, about 0.01% to about 2% of a lubricant. In someembodiments, the multi-component therapeutics, multi-component systems,and pharmaceutical compositions such as those described above may besuitable for oral administration, and in particular embodiments, themulti-component therapeutics, multi-component systems, andpharmaceutical compositions may be a solid oral dosage form.

In such formulations, the (R)-pramipexole may have a chiral purity of atleast 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least99.9%, preferably at least 99.95%, or at least 99.99%. In someembodiments, the chiral purity for (R)-pramipexole may be about 100%. Insome embodiments, of such formulations (R)-pramipexole may have a chiralpurity of 99.9% or greater, 99.95% or greater, or 99.99% or greater.

The amount of (R)-pramipexole in such formulations may be about 50 mg toabout 5,000 mg, from about 100 mg to about 3,000 mg, from about 300 mgto about 1,500 mg, from about 500 mg to about 1,000 mg. In someembodiments, the starting daily dose amount of (R)-pramipexole in theformulation may be about from about 25 mg to about 5,000 mg, from about50 mg to about 5,000 mg, from about 100 mg to about 5,000 mg, from about200 mg to about 5,000 mg, from about 250 mg to about 5,000 mg, fromabout 300 mg to about 5,000 mg, from about 400 mg to about 5,000 mg,from 450 mg to about 5,000 mg, from about 200 mg, to about 3,000 mg,from about 250 mg to about 3,000 mg, from about 300 mg to about 3,000mg, from about 400 mg to about 3,000 mg, from 450 mg to about 3,000 mg,from about 100 mg to about 1,000 mg, from about 200 mg to about 1,000mg, from about 250 mg to about 1,000 mg, from about 300 mg to about1,000 mg, from about 400 mg to about 1,000 mg, from about 600 mg toabout 1,000 mg, or from 450 mg to about 1,000 mg. In some embodiments ofthe formulation, (R)-pramipexole may be from about 600 mg to about 900mg. In some embodiments the multi-component therapeutics,multi-component systems, and pharmaceutical compositions of theinvention may include (R)-pramipexole having about 25 neuroprotectiveactivity equivalents and less than about 1.5 dopaminergic activityequivalents. In other embodiments, the multi-component therapeutics,multi-component systems, and pharmaceutical compositions of theinvention may have less than about 0.5 dopaminergic activity equivalentsor less than about 0.05 dopaminergic activity equivalents.

In some embodiments, the multi-component therapeutics, multi-componentsystems, and pharmaceutical compositions may have at least about 50, atleast about 75, at least about 125, at least about 150, at least about200, at least about 300, at least about 400, at least about 500, atleast about 750, at least about 750, or at least about 100neuroprotective activity equivalents. In some embodiments, themulti-component therapeutics, multi-component systems, andpharmaceutical compositions may have from about 50 to about 5,000, fromabout 100 to about 3,000, from about 300 to about 1,500, from about 500to about 1,000, from about 25 to about 5,000, from about 100 to about5,000, from about 200 to about 5,000, from about 250 to about 5,000,from about 300 to about 5,000, from about 400 to about 5,000, from 450to about 5,000, from about 200, to about 3,000, from about 250 to about3,000, from about 300 to about 3,000, from about 400 to about 3,000,from 450 to about 3,000, from about 100 to about 1,000, from about 200to about 1,000, from about 250 to about 1,000, from about 300 to about1,000, from about 400 to about 1,000, from about 600 to about 1,000,from 450 to about 1,000, or from about 600 to about 900 neuroprotectiveactivity equivalents.

The embodiments for the neuroprotective activity equivalents,dopaminergic activity equivalents, and dosage forms in themulti-component therapeutics, multi-component systems, andpharmaceutical compositions of the invention, which are described hereinseparately for the sake of brevity, can be joined in any suitablecombination.

Methods of Treatment, Uses, and Compositions and Compounds for Use

Embodiments of the invention further provide methods for treating aneurodegenerative disease by administering a therapeutically effectiveamount of (R)-pramipexole and a therapeutically effective amount of oneor more secondary agents. As such in some embodiments, (R)-pramipexoleand, in some embodiments, one or more secondary agents may be formulatedas a pharmaceutical or therapeutic composition by combining with one ormore pharmaceutically acceptable carriers. Embodiments includepharmaceutical or therapeutic compositions that may be administeredorally, preferably as a solid oral dose, and more preferably as a solidoral dose that may be a capsule or tablet. In a preferred embodiment,the pharmaceutical or therapeutic composition may be formulated intablet or capsule form for use in oral administration routes. Thecompositions and amounts of non-active ingredients in such a formulationmay depend on the amount of the active ingredient, and on the size andshape of the tablet or capsule. Such parameters may be readilyappreciated and understood by one of skill in the art. Thetherapeutically effective amount of (R)-pramipexole may be effective asan inhibitor of oxidative stress, an inhibitor of lipid peroxidation orin detoxification of oxygen radicals. Exemplary neurodegenerativedisorders which may be treated using the methods of various embodiments,include, for example, Parkinson' disease or symptoms thereof and ALS orsymptoms thereof. In one embodiment, Parkinson's disease and/or symptomsthereof are treated with the combination of (R)-pramipexole and one ormore secondary agents wherein the (R)-pramipexole and one or moresecondary agents may be administered in a single or multipleformulations.

For example, in various embodiments, a daily (R)-pramipexole may beadministered in combination with one or more secondary agents. Forexample, (R)-pramipexole may be administered to a patient in need oftreatment simultaneously with one or more secondary agents. In suchembodiments, the (R)-pramipexole and the one or more secondary agentsmay be combined in a single unit dosage form, or separate unit dosagesof (R)-pramipexole and the one or more secondary agents may beadministered simultaneously or within a relatively short amount of time.In other embodiments, (R)-primipexole may be administered alone in aunit dosage and one or more secondary agents may be administered in aseparate unit dosage at different times. For example, (R)-pramipexolemay be administered to a patient in need of treatment and a separateunit dose of one or more secondary agent may be administered one or morehour later than administration or (R)-prepexole. In some embodiments,(R)-pramipexole may be administered in combination with one or moresecondary agents and alone during the same course of treatment. Forexample, in particular embodiments, a unit dose of (R)-pramipexolecombined with one or more secondary agents may be administered, and atvarious other times throughout a 24 hour period, individual unit dosesof (R)-pramipexole or the one or more secondary agents may beadministered separately. Such embodiments may accommodate any treatmentschedule required to administer an effective daily dose of(R)-pramipexole and/or the one or more secondary agents in multiple ordivided doses.

The methods of various embodiments of the invention may constitute acourse of treatment and may be carried out for any length of time. Forexample, in some embodiments, a course of treatment may include aschedule of administrations wherein (R)-pramipexole and/or one or moresecondary therapeutic agents are administered one or more times in a24-hour period for, for example, 5 days or more. In other embodiments, acourse of treatment may include repeating administering (R)-pramipexoleand/or administering one or more secondary therapeutic agents one ormore times in a 24-hour period for, for example, 5 days to one or moreyears. In still other embodiments, a course of treatment includerepeated administration of (R)-pramipexole and/or one or more secondarytherapeutic agents may be carried out indefinitely, or for the lifetimeof the patient who is undergoing the course of treatment.

In certain embodiments, one course of treatment may be followed byanother course of treatment. For example, in some embodiments, a firstcourse of treatment in which (R)-pramipexole is administered in a firsttherapeutically effective amount and one or more secondary therapeuticagents administered at a therapeutically effective amount may be carriedout for a period of time such as, for example, 5 days to one or moremonths or one or more year. A second course of treatment may carried outa the conclusion of the first course of treatment, and the second courseof treatment may include, for example, a second therapeuticallyeffective amount of (R)-pramipexole that is different than the firsttherapeutically effective amount, a therapeutically effective amount ofone or more secondary therapeutic agents that is different than thetherapeutically effective amount administered in the first course oftreatment, one or more secondary therapeutic agents that are differentthan the one or more secondary therapeutic agents administered in thefirst course of treatment, or a combination thereof. Differences in thefirst and second courses of treatment may vary depending on the subjectto whom the courses of treatment is administered and may depend, forexample, on the subject's reaction to the treatment or changes in thesymptoms or severity of symptoms exhibit by the subject as a result ofthe treatment. The skilled artisan may observe such variations and alterthe course of treatment appropriately for each individual subject.

Further embodiments of the invention include methods of treatingneurodegenerative disorders or the symptoms associated withneurodegenerative disorders such as, for example, Parkinson's diseaseand/or ALS and methods of ameliorating the symptoms associated therewithusing the multi-component therapeutics, multi-component systems, andpharmaceutical compositions of the invention by administering(R)-pramipexole and one or more secondary therapeutic agents such as,for example, dopamine agonists, dopaminergic agonists, COMT inhibitors,MOA inhibitors, excitatory amino acid antagonists, growth factors,neurotrophic factors, antioxidants, anti-inflammatory agents,immunomodulators, anti-glutamatergics, ion channel blockers,α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptorantagonists, heat shock protein inducers/protein disaggregators anddownregulators, monoamine oxidase type B (MOAB) inhibitors, multi-targetagents, kinase inhibitors, Bcl inducers, histone deacetylase (HDAC)mediators, glial modulators, mitochondrial energy promoting agents,myostatin inhibitors, caspase inhibitors and combinations thereof.

Further embodiments of the invention are directed to methods ofdecreasing the rate of cell death associated with neurodegenerativedisorders such as, for example, Parkinson's disease and/or ALS using themulti-component therapeutics, multi-component systems, andpharmaceutical compositions of the invention by administering(R)-pramipexole and one or more secondary therapeutic agents such as,for example, dopamine agonists, dopaminergic agonists, COMT inhibitors,MOA inhibitors, excitatory amino acid antagonists, growth factors,neurotrophic factors, antioxidants, anti-inflammatory agents,immunomodulators, anti-glutamatergics, ion channel blockers,α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptorantagonists, heat shock protein inducers/protein disaggregators anddownregulators, monoamine oxidase type B (MOAB) inhibitors, multi-targetagents, kinase inhibitors, Bcl inducers, histone deacetylase (HDAC)mediators, glial modulators, mitochondrial energy promoting agents,myostatin inhibitors, caspase inhibitors and combinations thereof.

The (R)-pramipexole of such embodiments may have a chiral purity of atleast 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least99.9%, at least 99.95% and at least 99.99%, and in certain embodiments,the R-pramipexole may have a chiral purity of about 100%.

In some embodiments, a therapeutically effective amount of(R)-pramipexole may be administered to effectuate treatment and such atherapeutically effective amount may be from about 0.01 mg/kg/day toabout 10,000 mg/kg/day, from about 1 mg/kg/day to about 1,000 mg/kg/day,from about 0.1 mg/kg/day to about 1,000 mg/kg/day, from about 1mg/kg/day to about 1,000 mg/kg/day, from about 1,000 mg/kg/day to about10,000 mg/kg/day, or from about 1 mg/kg/day to about 100 mg/kg/day. Insome embodiments, the therapeutically effective amount of(R)-pramipexole may be from about 3 mg/kg/day to about 70 mg/kg/day. Insome embodiments, the therapeutically effective amount of(R)-pramipexole may be from about 7 mg/kg/day to about 40 mg/kg/day. Insome embodiment, the therapeutically effective amount of (R)-pramipexolemay be from about 3 mg/kg/day to about 50 mg/kg/day. In someembodiments, the dosage may be 10 mg/day to 1,500 mg/day, morepreferably 100 mg/day to 600 mg/day. In some embodiments, thetherapeutically effective amount of (R)-pramipexole may be from about 50mg to about 5,000 mg, from about 100 mg to about 3,000 mg, preferablyfrom about 300 mg to about 1,500 mg, or more preferably from about 500mg to about 1,000 mg. In some embodiments, the therapeutically effectiveamount of (R)-pramipexole may be from about 25 mg to about 5,000 mg,from about 50 mg to about 5,000 mg, from about 100 mg to about 5,000 mg,from about 200 mg to about 5,000 mg, from about 250 mg to about 5,000mg, from about 300 mg to about 5,000 mg, from about 400 mg to about5,000 mg, from 450 mg to about 5,000 mg, from about 200 mg to about3,000 mg, from about 250 mg to about 3,000 mg, from about 300 mg toabout 3,000 mg, from about 400 mg to about 3,000 mg, from 450 mg toabout 3,000 mg, from about 100 mg to about 1,000 mg, from about 200 mgto about 1,000 mg, from about 250 mg to about 1,000 mg, from about 300mg to about 1,000 mg, from about 400 mg to about 1,000 mg, from about600 mg to about 1,000 mg, or from 450 mg to about 1,000 mg. In someembodiments, the therapeutically effective amount of (R)-pramipexole isfrom about 600 mg to about 900 mg. This dose may be administered as asingle daily dose, or may be divided into several doses administeredthroughout the day, for example, 1 to 5 doses per day, preferably two tothree doses per day.

In some embodiments, the daily dose amount of (R)-pramipexole in suchmethods may be from about 0.01 mg/kg/day to about 10,000 mg/kg/day, fromabout 1 mg/kg/day to about 1,000 mg/kg/day, from about 0.1 mg/kg/day toabout 1,000 mg/kg/day, from about 1 mg/kg/day to about 1,000 mg/kg/day,from about 1,000 mg/kg/day to about 10,000 mg/kg/day, or from about 1mg/kg/day to about 100 mg/kg/day. In some embodiments, the daily doseamount of (R)-pramipexole may be from about 3 mg/kg/day to about 70mg/kg/day. In some embodiments, the daily dose amount of (R)-pramipexolemay be from about 7 mg/kg/day to about 40 mg/kg/day. In some embodiment,the daily dose amount of (R)-pramipexole may be from about 3 mg/kg/dayto about 50 mg/kg/day. In some embodiments, the daily dose amount may be10 mg/day to 1,500 mg/day, more preferably 100 mg/day to 600 mg/day. Insome embodiments, the daily dose amount of (R)-pramipexole is from about50 mg to about 5,000 mg, from about 100 mg to about 3,000 mg, from about300 mg to about 1,500 mg, or from about 500 mg to about 1,000 mg. Insome embodiments, the daily dose amount of (R)-pramipexole is from about25 mg to about 5,000 mg, from about 50 mg to about 5,000 mg, from about100 mg to about 5,000 mg, from about 200 mg to about 5,000 mg, fromabout 250 mg to about 5,000 mg, from about 300 mg to about 5,000 mg,from about 400 mg to about 5,000 mg, from 450 mg to about 5,000 mg, fromabout 200 mg to about 3,000 mg, from about 250 mg to about 3,000 mg,from about 300 mg to about 3,000 mg, from about 400 mg to about 3,000mg, from 450 mg to about 3,000 mg, from about 100 mg to about 1,000 mg,from about 200 mg to about 1,000 mg, from about 250 mg to about 1,000mg, from about 300 mg to about 1,000 mg, from about 400 mg to about1,000 mg, from about 600 mg to about 1,000 mg, or from 450 mg to about1,000 mg. In some embodiments, the daily dose amount of (R)-pramipexolemay be from about 600 mg to about 900 mg. In some embodiments, the dailydose amount may be from about 500 mg to about 1,000 mg of(R)-pramipexole. In some embodiments, daily dose amount may be fromabout 50 mg to about 5,000 mg of (R)-pramipexole. In some embodiments,the daily dose amount may be from about 100 mg to about 3,000 mg of(R)-pramipexole. In some embodiments, daily dose amount may be fromabout 200 mg to about 3,000 mg of (R)-pramipexole. In some embodiments,daily dose amount may be from about 300 mg to about 1,500 mg of(R)-pramipexole. In some embodiments, daily dose amount may be fromabout 500 mg to about 1,000 mg of (R)-pramipexole. This dose may beadministered as a single daily dose, or may be divided into severaldoses administered throughout the day, for example, 1 to 5 doses perday, preferably two to three doses per day.

In some embodiments, the daily dose amount may further include an amountof (S)-pramipexole that produces no observable adverse effect. In suchembodiments, a no observable effective dose amount of (S)-pramipexolemay be below 1.5 mg, below 0.5 mg, or below 0.05 mg per day. In otherembodiments, the daily dose amount may further include a non-effectivedose amount of (S)-pramipexole. In such embodiments, a non-effectivedose amount of (S)-pramipexole may be an amount not exceeding 1.0 mg perday. In other embodiments, the non-effective dose amount of(S)-pramipexole may be an amount that does not exceed 0.75 mg/day, 0.5mg/day, 0.25 mg/day, or 0.125 mg/day.

In some embodiments of the methods of the invention, the daily doseamount of (R)-pramipexole may be about 100 mg to about 3,000 mg and thechiral purity of the (R)-pramipexole may be 99.95% or greater. In otherembodiments, the daily dose amount of (R)-pramipexole may be about 200to about 3,000 mg and the chiral purity of the (R)-pramipexole may be99.95% or greater. In still other embodiments, the daily dose amount of(R)-pramipexole may be about 300 to about 1,500 mg and the chiral purityof the (R)-pramipexole may be 99.95% or greater. In yet otherembodiments of the methods, the daily dose amount of (R)-pramipexole maybe from about 500 mg to about 1,000 mg and the chiral purity of the(R)-pramipexole may be 99.95% or greater.

In further embodiments of the methods, the daily dose amount of(R)-pramipexole may be from about 100 mg to about 3,000 mg and the dailydose amount may include less than about 0.05 mg of (S)-pramipexole. Insome embodiments, the daily dose amount of (R)-pramipexole may be fromabout 200 mg to about 3,000 mg and the daily dose amount may includeless than about 0.05 mg of (S)-pramipexole. In other embodiments, thedaily dose amount of (R)-pramipexole may be from about 300 to about1,500 mg, and the daily dose amount may include less than about 0.05 mgof (S)-pramipexole. In yet other embodiments, the daily dose amount of(R)-pramipexole may be from about 500 mg to about 1,000 mg and the dailydose amount may include less than about 0.05 mg of (S)-pramipexole.

The embodiments for disease states, patient type (naïve vs. not naïve),daily dose amounts, no observable adverse effect level dose amounts,non-effective dose amounts, and chiral purities for the methods of theinvention, which are described herein separately for the sake ofbrevity, can be joined in any suitable combination.

Kits

Some embodiments of the invention include kits that may include themulti-component therapeutics, multi-component systems, andpharmaceutical compositions of the invention including (R)-pramipexoleand one or more secondary therapeutic agents such as, for example,dopamine agonists, dopaminergic agonists, COMT inhibitors, MOAinhibitors, excitatory amino acid antagonists, growth factors,neurotrophic factors, antioxidants, anti-inflammatory agents,immunomodulators, anti-glutamatergics, ion channel blockers,α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptorantagonists, heat shock protein inducers/protein disaggregators anddownregulators, monoamine oxidase type B (MOAB) inhibitors, multi-targetagents, kinase inhibitors, Bcl inducers, histone deacetylase (HDAC)mediators, glial modulators, mitochondrial energy promoting agents,myostatin inhibitors, caspase inhibitors and combinations thereof, andinstructions for administering or prescribing the one or morepharmaceutical compositions. In such embodiments, a direction may beincluded to administer or prescribe to a patient the multi-componenttherapeutics, multi-component systems, and pharmaceutical compositionsof the invention at a starting daily dose of at least about 50 mg toabout 5,000 mg of (R)-pramipexole and a therapeutically effective amountof one or more second therapeutic agents. In addition, in someembodiments, kits may include one or more pharmaceutical compositionssuch as those described in any of the embodiments of the compositionsdescribed herein, or any combination thereof, and instructions foradministering or prescribing to a patient the one or more pharmaceuticalcompositions such as, for example, a direction to administer orprescribe the one or more pharmaceutical compositions or any combinationthereof.

The (R)-pramipexole for use in the kits of the invention may have achiral purity of at least 99.5%, preferably at least 99.6%, preferablyat least 99.7%, preferably at least 99.8%, preferably at least 99.9%,preferably at least 99.95% and more preferably at least 99.99%. Inparticular embodiments, the chiral purity for the R-pramipexole may be100%. In other embodiments, (R)-pramipexole may have a chiral purity of99.9% or greater, 99.95% or greater, or 99.99% or greater.

In some embodiments, the instructions may include an instruction toadminister or prescribe to a patient in need of treatment themulti-component therapeutics, multi-component systems, andpharmaceutical compositions of the invention in accordance with theembodiments described above at a starting daily dose of (R)-pramipexoleof from about 0.1 mg/kg/day to about 1,000 mg/kg/day or from about 1mg/kg/day to about 100 mg/kg/day. In some embodiments, the instructionsmay include an instruction to administer or prescribe to a patient themulti-component therapeutics, multi-component systems, andpharmaceutical compositions of the invention at a starting daily dose of(R)-pramipexole of from about 3 mg/kg/day to about 70 mg/kg/day or fromabout 7 mg/kg/day to about 40 mg/kg/day. In other embodiments, theinstructions may include an instruction to administer or prescribe to apatient the multi-component therapeutics, multi-component systems, andpharmaceutical compositions of the invention at a starting daily dose of(R)-pramipexole of from about 50 mg to about 5,000 mg, from about 100 mgto about 3,000 mg, from about 300 mg to about 1,500 mg, or from about500 mg to about 1,000 mg. In some embodiments, the instructions mayinclude a direction to administer or prescribe to a patient themulti-component therapeutics, multi-component systems, andpharmaceutical compositions of the invention at a starting daily dose of(R)-pramipexole of from about 25 mg to about 5,000 mg, from about 50 mgto about 5,000 mg, from about 100 mg to about 5,000 mg, from about 200mg to about 5,000 mg, from about 250 mg to about 5,000 mg, from about300 mg to about 5,000 mg, from about 400 mg to about 5,000 mg, from 450mg to about 5,000 mg, from about 200 mg to about 3,000 mg, from about250 mg to about 3,000 mg, from about 300 mg to about 3,000 mg, fromabout 400 mg to about 3,000 mg, from 450 mg to about 3,000 mg, fromabout 100 mg to about 1,000 mg, from about 200 mg to about 1,000 mg,from about 250 mg to about 1,000 mg, from about 300 mg to about 1,000mg, from about 400 mg to about 1,000 mg, from about 600 mg to about1,000 mg, or from 450 mg to about 1,000 mg. In some embodiments, thestarting daily dose amount of (R)-pramipexole may be from about 600 mgto about 900 mg. The doses described above may be administered as asingle daily dose, or may be divided into several doses administeredthroughout the day, for example, 1 to 5 doses per day, preferably two tothree doses per day, and the instructions of various embodiments maydescribe such divided administrations.

In some embodiments, the instructions comprise a direction to administeror prescribe the multi-component therapeutics, multi-component systems,and pharmaceutical compositions of the invention in an amount sufficientto result in administration of a starting daily dose of from about 100mg to about 3,000 mg of (R)-pramipexole to a patient. In someembodiments, instructions may include a direction to administer orprescribe the multi-component therapeutics, multi-component systems, andpharmaceutical compositions of the invention in an amount sufficient toresult in administration of a starting daily dose of from about 200 mgto about 3,000 mg of (R)-pramipexole to a patient. In some embodiments,the instructions comprise a direction to administer or prescribe themulti-component therapeutics, multi-component systems, andpharmaceutical compositions of the invention in an amount sufficient toresult in administration of from about 300 to about 1,500 mg of(R)-pramipexole to a patient. In some embodiments, the instructionscomprise a direction to administer or prescribe the multi-componenttherapeutics, multi-component systems, and pharmaceutical compositionsof the invention in an amount sufficient to result in administration ofa starting daily dose of from about 500 to about 1,000 mg of(R)-pramipexole to a patient.

The multi-component therapeutics, multi-component systems, andpharmaceutical compositions of the invention of the invention may beprepared, packaged, sold in bulk, as a single unit dose, or as multipleunit doses. The various compositions associated with the multi-componenttherapeutics, multi-component systems, and pharmaceutical compositionsof the invention may be formulated to be administered orally,ophthalmically, intravenously, intramuscularly, intra-arterially,intramedularry, intrathecally, intraventricularly, transdermally,subcutaneously, intraperitoneally, intravesicularly, intranasally,enterally, topically, sublingually, or rectally. The variouscompositions of the invention may be administered orally, preferably asa solid oral dose, and more preferably as a solid oral dose that may bea capsule or tablet. In some embodiments, the compositions of theinvention may be formulated as tablets for oral administration.

The multi-component therapeutics, multi-component systems, andpharmaceutical compositions of the invention can be administered in theconventional manner by any route where they are active. Administrationcan be systemic, topical, or oral. For example, administration can be,but is not limited to, parenteral, subcutaneous, intravenous,intramuscular, intraperitoneal, transdermal, oral, buccal, or ocularroutes, or intravaginally, intravesicularly, by inhalation, by depotinjections, or by implants. Thus, modes of administration for thecompounds of the present invention (either alone or in combination withother pharmaceuticals) can be, but are not limited to, sublingual,injectable (including short-acting, depot, implant and pellet formsinjected subcutaneously or intramuscularly), or by use of vaginalcreams, suppositories, pessaries, vaginal rings, rectal suppositories,intrauterine devices, and transdermal forms such as patches and creams.

The doses of the (R)-pramipexole and one or more second therapeuticagents which may be administered to a patient in need thereof in themulti-component therapeutics, multi-component systems, andpharmaceutical compositions of the invention may range between about 0.1mg/kg per day and about 1,000 mg/kg per day. This dose may beadministered as a single daily dose, or may be divided into severaldoses which are administered throughout the day, such as 1 to 5 doses,or two to three doses per day. The route of administration may includeoral, sublingual, transdermal, rectal, or any accessible parenteralroute. One of ordinary skill in the art will understand and appreciatethe dosages and timing of the dosages to be administered to a patient inneed thereof. The doses and duration of treatment may vary, and may bebased on assessment by one of ordinary skill in the art based onmonitoring and measuring improvements in neuronal and non-neuronaltissues. This assessment may be made based on outward physical signs ofimprovement, such as increased muscle control, or on internalphysiological signs or markers. The doses may also depend on thecondition or disease being treated, the degree of the condition ordisease being treated and further on the age and weight of the patient.

Specific modes of administration will depend on the indication. Theselection of the specific route of administration and the dose regimenor course of treatment may be adjusted or titrated by the clinicianaccording to methods known to the clinician in order to obtain theoptimal clinical response. The amount of compound to be administered maybe that amount which is therapeutically effective. The dosage to beadministered may depend on the characteristics of the subject beingtreated, e.g., the particular animal or human subject treated, age,weight, health, types of concurrent treatment, if any, and frequency oftreatments, and can be easily determined by one of skill in the art(e.g., by the clinician).

Pharmaceutical formulations containing the various compounds of theinvention and a suitable carrier may also be any number of solid dosageforms which include, but are not limited to, tablets, capsules, cachets,pellets, pills, powders and granules; topical dosage forms whichinclude, but are not limited to, solutions, powders, fluid emulsions,fluid suspensions, semi-solids, ointments, pastes, creams, gels andjellies, and foams; and parenteral dosage forms which include, but arenot limited to, solutions, suspensions, emulsions, and dry powder;comprising an effective amount of a polymer or copolymer of the presentinvention. It is also known in the art that the active ingredients canbe contained in such formulations with pharmaceutically acceptablediluents, fillers, disintegrants, binders, lubricants, surfactants,hydrophobic vehicles, water soluble vehicles, emulsifiers, buffers,humectants, moisturizers, solubilizers, preservatives and the like. Themeans and methods for administration are known in the art and an artisancan refer to various pharmacologic references for guidance. For example,Modern Pharmaceutics, Banker & Rhodes, Marcel Dekker, Inc. (1979); andGoodman & Gilman's The Pharmaceutical Basis of Therapeutics, 6thEdition, MacMillan Publishing Co., New York (1980) can be consulted.

In particular embodiments, the route of administration of thecompositions of the invention may be oral, with a more preferable routebeing in the form of tablets, capsules, lozenges and the like. In suchembodiments, the compositions of the present invention may be formulatedas tablets for oral administration. A tablet may be made by compressionor molding, optionally with one or more accessory ingredients.Compressed tablets may be prepared by compressing in a suitable machinethe active ingredient in a free-flowing form such as a powder orgranules, optionally mixed with a binder, lubricant, inert diluent,lubricating, surface active or dispersing agent. Molded tablets may bemade by molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets may be uncoated or they may be coated by known techniques,optionally to delay disintegration and absorption in thegastrointestinal tract and thereby providing a sustained action over alonger period. The coating may be adapted to release the active compoundin a predetermined pattern (e.g., in order to achieve a controlledrelease formulation) or it may be adapted not to release the activecompound until after passage of the stomach (enteric coating). Thecoating may be a sugar coating, a film coating (e.g., based onhydroxypropyl methylcellulose, methylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, carboxymethyl cellulose,acrylate copolymers, polyethylene glycols and/or polyvinylpyrrolidone),or an enteric coating (e.g., based on methacrylic acid copolymer,cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate,hydroxypropyl methylcellulose acetate succinate, polyvinyl acetatephthalate, shellac, and/or ethylcellulose). Furthermore, a time delaymaterial such as, e.g., glyceryl monostearate or glyceryl distearate maybe employed. The solid tablet compositions may include a coating adaptedto protect the composition from unwanted chemical changes, (e.g.,chemical degradation prior to the release of the active drug substance).

For oral administration, the compounds can be formulated readily bycombining these compounds with pharmaceutically acceptable carriers wellknown in the art. Such carriers enable the compounds of the invention tobe formulated as tablets, pills, dragees, capsules, liquids, gels,syrups, slurries, suspensions and the like, for oral ingestion by apatient to be treated. Pharmaceutical preparations for oral use can beobtained by adding a solid excipient, optionally grinding the resultingmixture, and processing the mixture of granules, after adding suitableauxiliaries, if desired, to obtain tablets or dragee cores. Suitableexcipients include, but are not limited to, fillers such as sugars,including, but not limited to, lactose, sucrose, mannitol, and sorbitol;cellulose preparations such as, but not limited to, maize starch, wheatstarch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,and polyvinylpyrrolidone (PVP). If desired, disintegrating agents can beadded, such as, but not limited to, the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate.

Dragee cores can be provided with suitable coatings. For this purpose,concentrated sugar solutions can be used, which can optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments can be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include, but arenot limited to, push-fit capsules made of gelatin, as well as soft,sealed capsules made of gelatin and a plasticizer, such as glycerol orsorbitol. The push-fit capsules can contain the active ingredients inadmixture with filler such as, e.g., lactose, binders such as, e.g.,starches, and/or lubricants such as, e.g., talc or magnesium stearateand, optionally, stabilizers. In soft capsules, the active compounds canbe dissolved or suspended in suitable liquids, such as fatty oils,liquid paraffin, or liquid polyethylene glycols. In addition,stabilizers can be added. All formulations for oral administrationshould be in dosages suitable for such administration.

For buccal or sublingual administration, the compositions can take theform of tablets, flash melts or lozenges formulated in any conventionalmanner.

The compounds of the present invention can be formulated for parenteraladministration by injection, e.g., by bolus injection or continuousinfusion. The compounds can be administered by continuous infusion overa period of about 15 minutes to about 24 hours. Formulations forinjection can be presented in unit dosage form, e.g., in ampoules or inmulti-dose containers, with an added preservative. The compositions cantake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitcan be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator can be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds of the present invention can also be formulated in rectalcompositions such as suppositories or retention enemas, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds ofthe present invention can also be formulated as a depot preparation.Such long acting formulations can be administered by implantation (forexample subcutaneously or intramuscularly) or by intramuscularinjection. Depot injections can be administered at about 1 to about 6months or longer intervals. Thus, for example, the compounds can beformulated with suitable polymeric or hydrophobic materials (forexample, as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

In transdermal administration, the compounds of the present invention,for example, can be applied to a plaster, or can be applied bytransdermal, therapeutic systems that are consequently supplied to theorganism.

Pharmaceutical and therapeutic compositions of the compounds also cancomprise suitable solid or gel phase carriers or excipients. Examples ofsuch carriers or excipients include but are not limited to calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin, and polymers such as, e.g., polyethylene glycols.

The compounds of the present invention can also be administered incombination with other active ingredients, such as, for example,adjuvants, protease inhibitors, or other compatible drugs or compoundswhere such combination is seen to be desirable or advantageous inachieving the desired effects of the methods described herein.

Preparation of (R)- and (S)-Pramipexole

Processes for the preparation of pramipexole are described in U.S. Pat.No. 4,843,086 and U.S. Pat. No. 4,886,812 to Griss et al., each of whichis incorporated by reference in its entirety. The (R)-pramipexole of thepresent invention may be synthesized and/or purified by methodsdisclosed in the copending U.S. Provisional Application No. 60/894,829entitled “Methods of Synthesizing and Purifying R(+) and(S)-pramipexole”, filed on Mar. 14, 2007, and U.S. ProvisionalApplication No. 60/894,814 entitled “Methods of EnantiomericallyPurifying Chiral Compounds”, filed on Mar. 14, 2007, which areincorporated herein by reference in their entireties. Specifically,preparations of pramipexole which are chirally pure for the R(+)enantiomer may be produced using a bi-molecular nucleophilicsubstitution (S_(N)2) reaction. A diamine, 2,6diamino-4,5,6,7-tetrahydro-benzothiazole, is reacted with a propylsulfonate or a propyl halide in polar solvents to generate an insolublepramipexole salt in a one pot synthesis scheme. The pramipexole saltreaction product displays a high chemical purity and an increasedoptical purity over the reactants, which may be due to limitedsolubility of the pramipexole salt in the polar solvents of the reactionmixture. Purification of the final pramipexole synthesis product fromthe reaction mixture thus involves simple trituration and washing of theprecipitated pramipexole salt in a volatile solvent such as an alcoholor heptane, followed by vacuum drying.

In some embodiments, the (R)-pramipexole is prepared by dissolving adiamine of formula 2,6 diamino-4,5,6,7-tetrahydro-benzothiazole in anorganic solvent, reacting the diamine with a propyl sulfonate or apropyl halide under conditions sufficient to generate and precipitatethe pramipexole salt, and recovering the pramipexole salt. In apreferred embodiment, the propyl sulfonate may be propyl tosylate. In afurther embodiment, the propyl halide may be propyl bromide. Thepramipexole salt reaction product of this process displays a highchemical purity and an increased optical purity over the reactants.Without wishing to be bound by theory, the increased optical purity maybe due to limited solubility of the pramipexole salt reaction product inthe polar solvents of the reaction mixture. Purification of the finalpramipexole reaction product from the reaction mixture thus involvessimple trituration and washing of the precipitated pramipexole salt in avolatile solvent such as an alcohol or heptane, followed by vacuumdrying.

In embodiments of the process, the diamine may be an R(+) diamine, or amixture of the R(+) and an S diamine. The chemical purity of the finalpramipexole salt may be at least about 97% or greater, preferably 98% orgreater, more preferably 99% or greater. The R(+) enantiomers of thepramipexole salt generated using this process are generated fromstarting diamines which may be at least 55% optically pure, preferably70% optically pure, and more preferably greater than 90% optically pure.The final pramipexole product may be enriched to 99.6% optical purity orgreater, 99.7% optical purity or greater, preferably 99.8% opticallypurity or greater, and more preferably 99.9% optical purity or greater,99.95% optical purity or greater, 99.99% optical purity or greater. Insome embodiments, the optical purity may be 100%.

In embodiments of the process, the organic solvent may be a polaraprotic solvent such as tetrahydofuran, dimethylformamide, dimethylsulfoxide, dimethylacetamide, or hexamethylphosphoric triamide. Theorganic solvent may also be a low molecular weight alcohol such asethanol, 1-propanol, or n-butanol. Further, the organic solvent may beany combination of the polar aprotic solvents and low molecular weightalcohols. The organic solvent may have a water content of from about 0to about 10 volume percent. Preferably, the solvents used in thepractice of this invention are standard ACS grade solvents. Further, thepropyl sulfonate or a propyl halide may be added at about 1.0 to about2.0 molar equivalents of the diamine.

In further embodiments of the process, the conditions sufficient togenerate and precipitate the pramipexole salt may comprise heating thedissolved diamine at an elevated temperature, adding the propylsulfonate or propyl halide which may be dissolved indi-isoproplyethylamine and an organic solvent to form a mixture, andstirring the mixture for about 4 hours. Alternatively, thedi-isoproplyethylamine may be added to the reaction with the diamine,and the propyl sulfonate or propyl halide may be dissolved in an organicsolvent to form a mixture, which may be added to the reaction withstirring over about 4 hours.

In this embodiment, the elevated temperature of the reaction may bebelow the boiling temperature of the reaction, specifically, below theboiling temperature of the organic solvent(s) of the reaction mixture.The elevated temperature may be lower than about 125° C., preferablylower than about 100° C., and more preferably about 95° C. or lower. Thetimes necessary for the reaction may vary with the identities of thereactants, the solvent system and with the chosen temperature.

In an alternative embodiment, the conditions sufficient to generate andprecipitate the pramipexole salt may comprise using dimethylformamide asthe organic solvent, heating the dissolved diamine at an elevatedtemperature, adding the propyl sulfonate or propyl halide which isdissolved in dimethylformamide to form a mixture, and stirring themixture for about 4 hours. The elevated temperature of the reaction maybe below the boiling temperature of the reaction, specifically, belowthe boiling temperature of the organic solvent(s) of the reactionmixture. The elevated temperature may be lower than about 125° C.,preferably lower than about 100° C., and more preferably about 75° C. orlower. The times necessary for the reaction may vary with the identitiesof the reactants, the solvent system and with the chosen temperature.

In a preferred alternative embodiment, the conditions sufficient togenerate and precipitate the pramipexole salt comprise usingdimethylformamide as the organic solvent and heating the dissolveddiamine at an elevated temperature. A mixture of propyl sulfonate orpropyl halide, at preferably 1.25 molar equivalents, dissolved indimethylformamide, preferably 10 volumes, and di-isoproplyethylamine,preferably 1.25 molar equivalents, is added slowly to the heated diaminewith stirring over a period of about 4 hours. Alternatively, thedi-isoproplyethylamine may be added to the reaction with the diamine,and the propyl sulfonate or propyl halide may be dissolved indimethylformamide to form a mixture, which may be added to the reactionwith stirring for about 4 hours. The elevated temperature of thereaction may be below the boiling temperature of the reaction,specifically, below the boiling temperature of the organic solvent(s) ofthe reaction mixture. The elevated temperature may be lower than about125° C., preferably lower than about 100° C., and more preferably about65° C. or lower. The times necessary for the reaction may vary with theidentities of the reactants, the solvent system and with the chosentemperature.

Embodiments of the process further comprise cooling the reaction to atemperature of about room temperature, about 25° C., and stirring thereaction for about 2 hours. The process may further involve filteringthe reaction to isolate a solid precipitate, washing the precipitatewith an alcohol, and drying the precipitate under vacuum. Thepramipexole salt reaction product of this process may display anincreased optical purity over the reactants.

Alternatively, the pramipexole sulfonate or halide salt can be reactedwith concentrated HCl in an organic solvent, such as an alcohol, at atemperature of from about 0 to about 5° C. An organic solvent, such asmethyl tert-butyl ether (MTBE), may be added, and the reaction may bestirred for an additional hour. The (R)-pramipexole dihydrochlorideproduct may be recovered from the reaction mixture by filtering, washingwith an alcohol and vacuum drying.

In an embodiment of the process, referred to as condition A in Table 7and in the examples, the reaction condition which may be sufficient togenerate the pramipexole product may include heating the dissolveddiamine of formula II to an elevated temperature with continuousstirring. The elevated temperature is preferably less than the meltingpoint of the chosen organic solvent, lower than about 125° C.,preferably lower than about 100° C., and more preferably about 95° C. Asolution of propyl sulfonate or propyl halide, which is dissolved indi-isoproplyethylamine and an organic solvent to form a mixture, isadded slowly over a period of several hours. This reaction mixture maythen be stirred at temperature for an additional period of time such as,for example, about 4 hours. The times necessary for the reaction mayvary with the identities of the reactants and solvent system, and withthe chosen temperature, and would be understood by one of skill in theart.

In an alternate embodiment, the di-isoproplyethylamine may be added tothe reaction with the diamine, and the propyl sulfonate or propyl halidemay be dissolved in an organic solvent to form a mixture, which may beadded to the reaction with stirring over a period of several hours. Thisreaction mixture may then be stirred at temperature for an additionalperiod of time such as, for example, at least 4 hours. The timenecessary for the reaction to run to completion may vary with theidentities of the reactants and solvent system, and with the chosentemperature, and would be understood by one of skill in the art.

In an alternative embodiment of the process, referred to as condition Bin Table 7, the reaction conditions which are sufficient to generate thepramipexole product may include using dimethylformamide as the organicsolvent, and heating the dissolved diamine of formula II to an elevatedtemperature with continuous stirring. The elevated temperature ispreferably less than the melting point of the chosen organic solvent,lower than about 125° C., preferably lower than about 100° C., and morepreferably about 75° C. A solution of propyl sulfonate or propyl halide,which is dissolved in dimethylformamide, may be added slowly over aperiod of several hours. This reaction mixture may then be stirred attemperature for an additional period of time such as, for example, about4 hours. The time necessary for the reaction to run to completion mayvary with the identities of the reactants and solvent system, and withthe chosen temperature, and would be understood by one of skill in theart.

In a preferred alternative embodiment of the process, referred to ascondition C in Table 7, the reaction includes using dimethylformamide asthe organic solvent for dissolution of the diamine. The diamine offormula II may then be heated to an elevated temperature with continuousstirring. The elevated temperature is preferably less than the meltingpoint of the chosen organic solvent, lower than about 125° C.,preferably lower than about 100° C., and more preferably about 65° C. Asolution of propyl sulfonate or propyl halide, preferably about 1.25molar equivalents, may be dissolved in dimethylformamide, preferablyabout 10 volumes, and di-isoproplyethylamine, preferably about 1.25molar equivalents, to form a mixture. This mixture may be added slowlyover a period of several hours to the heated diamine. This reactionmixture may then be stirred at temperature for an additional period oftime such as, for example, about 4 hours. Alternatively, thedi-isoproplyethylamine may be added to the reaction with the diamine,and the propyl sulfonate or propyl halide may be dissolved indimethylformamide to form a mixture, which may be added to the reactionwith stirring over a period of several hours. This reaction mixture maythen be stirred at temperature for an additional period of time such as,for example, about 4 hours. The time necessary for the reaction to runto completion may vary with the identities of the reactants and solventsystem, and with the chosen temperature, and would be understood by oneof skill in the art.

Purification of the final pramipexole product may include cooling thereactions disclosed above to a temperature of about 25° C., and stirringthe reactions for a period of time such as, for example, about 2 hours.The purification may further include filtering the reaction to isolate asolid precipitate, washing the precipitate with an alcohol, and dryingthe precipitate under vacuum. The final products of the reaction may beanalyzed by high pressure liquid chromatography (HPLC) for chemical andchiral purity.

Further, ¹H NMR and ¹³C NMR may be used to confirm the structure of theproduct pramipexole. Results of example syntheses using each of theseveral conditions which are embodiments of the present disclosure arelisted in Table 7. Several example syntheses of pramipexole usingconditions A and C of the present disclosure are detailed in Examples5-7.

The sulfonate or halide salts of pramipexole may be converted to an HClsalt using a concentrated solution of HCl in ethanol. A p-TSApramipexole salt may be re-dissolved in an alcohol, such as ethanol, andthe mixture may be cooled to between about 0 and about 5° C. withcontinuous stirring. A concentrated HCl may then be added, followed by asolvent such as methyl tert-butyl ether (MTBE), and the mixture may bestirred for an hour at between about 0 and about 5° C. The reactionmixture may then be filtered, washed with an MTBE/alcohol solution, anddried under vacuum. The final product is pramipexole dihydrochloride. Adetailed example of this synthesis may be found in Example 8.

An alternate method for conversion of the sulfonate or halide salts ofpramipexole to an HCl salt involves the use of a concentrated solutionof HCl and isopropyl acetate (IPAC). A sulfonate or halide salt ofpramipexole may be taken up in IPAC and cooled to 15° C. HCl (gas) maybe bubbled into the slurry for about 1 hour, after which the mixture maybe filtered, washed with IPAC and dried under vacuum at room temperatureto afford a pramipexole dihydrochloride salt. A detailed example of thissynthesis may be found in Example 9.

The sulfonate or halide salts of pramipexole may alternatively beconverted to the free base form of pramipexole. A p-TSA pramipexole saltmay be dissolved in dichloromethane (DCM) and water. The resultingsolution may then by brought to a pH of about 11-12 using NaOH. Twophases may be generated, and the aqueous phase may be extracted withDCM, dried over magnesium sulfate (MgSO₄), filtered over Celite® andconcentrated. The concentrated residue may be re-dissolved in MTBE andstirred as a slurry for several hours. The solids may then be filtered,washed with MTBE, and dried under vacuum at a temperature of about 35°C. The final product is pramipexole free base. A detailed example ofthis synthesis may be found in Example 10.

Alternatively, the sulfonate or halide salts of pramipexole mayalternatively be converted to the free base form of pramipexole by asecond process. In this second process, the p-TSA salt of pramipexole isdissolved in water and cooled to a temperature of about 10° C. Thisslurry is basified by addition of NaOH, diluted with brine, andextracted several times in DCM. The combined organic phases are thenwashed with brine, dried over MgSO₄, filtered and concentrated todryness. A detailed example of this synthesis may be found in Example11.

The free base form of pramipexole may be converted to pramipexoledihydrochloride by bubbling HCl gas into a cooled solution of thepramipexole free base in IPAC. Alternatively, the free base form ofpramipexole may be converted to pramipexole dihydrochloride by mixingwith concentrated HCl at room temperature overnight. Detailed examplesof the aforementioned synthesis schemes may be found in Examples 12 and13, respectively.

Alternatively, the free base form of pramipexole may be converted topramipexole fumarate by the addition of 2 molar equivalents of fumaricacid.

TABLE 7 Experiments for S_(N)2 preparation of pramipexole pTSA saltCondition Isomer Batch Size Results A R(+) 45 grams Yield = 53.2 grams(52%) Chemical Purity = 98.2% AUC by HPLC Chiral Purity = >99.5% AUC byHPLC A S(−) 5 grams Yield = 4.99 grams (44.2%) Chemical Purity = 98.0%AUC by HPLC Chiral Purity = >99.6% AUC by HPLC A Racemic 5 gram Yield =5.12 grams (45%) Chemical Purity = 97.1% AUC by HPLC Chiral Purity = 1:1R(+):S(−) by HPLC B R(+) 5 gram Yield = 4.6 grams (40%) Chemical Purity= 94.9% AUC by HPLC Chiral Purity = 99.6% AUC by HPLC B S(−) 10 gramYield = 9.81 grams (43.3%) Chemical Purity = 94.9% AUC by HPLC ChiralPurity = 99.7% AUC by HPLC B Racemic 5 gram Yield = 2.9 grams (25.6%)Chemical Purity = 98.3% AUC by HPLC Chiral Purity = 1:1 R(+):S(−) byHPLC C R(+) 250 gram Yield = 317.6 grams (56%) Chemical Purity = 99.4%AUC by HPLC Chiral Purity = 99.8% AUC by HPLC C S(−) 20 gram Yield =25.41 grams (56%) Chemical Purity = 99.4% AUC by HPLC Chiral Purity =99.7% AUC by HPLC C Racemic 5 gram Yield = 6.02 grams (53.1%) ChemicalPurity = 99.2% AUC by HPLC Chiral Purity = 1:1 R(+):S(−) by HPLC E* R(+)25 gram Yield = 47% Chiral Purity = 99.8% AUC by HPLC E* S(−) 25 gramYield = 47% Chiral Purity = 99.8% AUC by HPLC *Condition E is the sameas Condition C, except that the recovery step does not incorporatedilution in MTBE. The MTBE increases the recovery (yield) from thesynthesis reaction, but may reduce the overall chiral purity. ConditionE is explained in more detail in Table 9.

The alternative process for preparing an enantiomerically purepramipexole from a mixture of (R)-pramipexole and (S)-pramipexoleinvolves using acid addition and trituration (precipitation) of anenantiomerically pure pramipexole based on insolubility of theenantiomers (R(+) and S(−)) in the resulting achiral salt solution. Inembodiments of this process, enantiomerically pure pramipexole istriturated from an acid addition solution based on the insolubility ofthe enantiomers in the resulting achiral salt reagents. This embodiment,a process for preparing an enantiomerically pure pramipexole, comprisesdissolving an enantiomerically enriched pramipexole in an organicsolvent at an elevated temperature, adding a selected acid, cooling thereaction to room temperature, stirring the cooled reaction at roomtemperature for an extended time and recovering enantiomerically pure(R)-pramipexole. In a preferred embodiment, the selected acid may beadded at from about 1 molar equivalent to about 2 molar equivalents ofthe enantiomerically enriched pramipexole.

In an embodiment of the process, the selected acid is p-toluenesulfonicacid (p-TSA) and the organic solvent is ethanol. In another embodimentof the process, the elevated temperature may be from about 65° C. toabout 85° C. and the cooling occurs at a rate of about 25° C. per hour.The elevated temperature may also be a temperature lower than 125° C.,preferably lower than 100° C., and more preferably about 75° C. Thetimes necessary for the reaction may vary with the identities of thereactants, the solvent system and with the chosen temperature, and maybe easily appreciated by one of skill in the art. In yet anotherembodiment of the process, recovering enantiomerically pure pramipexolecomprises cooling the reaction to a temperature of about 25° C. andstirring the reaction for at least about 2 hours. The recovery mayfurther comprise filtering the reaction to isolate a solid precipitate,washing the precipitate with an alcohol and drying the precipitate undervacuum.

In various embodiments of the process, the organic solvent may include,but is not limited to, acetonitrile, acetone, ethanol, ethyl acetate,methyl tert-butyl ether, methyl ethyl ketone, isopropyl acetate andisopropyl alcohol. In a preferred embodiment, the organic solvent isethanol. The acid may include, but is not limited to, halogenic acidssuch as, for example, hydrobromic, hydrochloric, hydrofluoric andhydroiodic acid; inorganic acids such as, for example, nitric,perchloric, sulfuric and phosphoric acid; organic acids such as, forexample, sulfonic acids (methanesulfonic, trifluoromethan sulfonic,ethanesulfonic, benzenesulfonic or p-toluenesulfonic), acetic, malic,fumaric, succinic, citric, benzoic, gluconic, lactic, mandelic, mucic,pamoic, pantothenic, oxalic and maleic acid; and aminoacids such asaspartic or glutamic acid. The acid may be a mono- or di-acid, such as adi-hydrohalogenic, di-sulfuric, di-phosphoric or di-organic acid. In allcases, the acid is used as an achiral reagent which is not selected onthe basis of any expected or known preference for interaction with orprecipitation of a specific optical isomer of the products of thisdisclosure. In a preferred embodiment, the selected acid isp-toluenesulfonic acid.

In embodiments of the process, the final chiral purity for an R(+)enantiomer of the pramipexole salt may be greater than 99% when thestarting mixture contains pramipexole which is at least 55% opticallypure for the R(+) enantiomer, preferably 80% optically pure for the R(+)enantiomer, preferably 85% optically pure for the R(+) enantiomer, morepreferably 90% optically pure for the R(+) enantiomer and mostpreferably 95% optically pure for the R(+) enantiomer. The final chiralpurity for an S(−) enantiomer of the pramipexole salt may be greaterthan 99% when the starting mixture contains pramipexole which is atleast 55% optically pure for the S(−) enantiomer, preferably 80%optically pure for the S(−) enantiomer, preferably 85% optically purefor the S(−) enantiomer, more preferably 90% optically pure for the S(−)enantiomer and most preferably 95% optically pure for the S(−)enantiomer. The chiral purity of the final pramipexole salt maypreferably be 99.6% or greater, 99.7% or greater, preferably 99.8% orgreater, and more preferably 99.9% or greater. In some embodiments, thechiral purity of the final pramipexole salt may be 100%.

In embodiments, after the enantiomerically enriched pramipexole isdissolved in an organic solvent at an elevated temperature and the acidis added, the reaction may be cooled to room temperature at a rate ofabout 25° C./hour. The enantiomerically pure pramipexole may then berecovered from the reaction solution by stirring the reaction for atleast about 2 hours, filtering the reaction to isolate a solidprecipitate, washing the precipitate with an alcohol and drying theprecipitate under vacuum. The rates of cooling and the time required forthe additional stirring may vary with the chosen organic solvent andacid, and may be easily appreciated by one skilled in the art.Additionally, the reaction volumes may dictate the degree of opticalpurification and the overall yield of the final pramipexole product.These volumes would be understood and appreciated by one of skill in theart. Examples of specific times, temperatures and volumes which enablethe practice of this invention are given in the Examples.

In embodiments, the chiral purity of the pramipexole salt product forthe R(+) enantiomer may be greater than 99% when the chiral purity ofthe starting pramipexole mixture for the R(+) enantiomer is greater than55%, preferably greater than 70%, or more preferably greater than 90%.The chiral purity of the final pramipexole salt may be 99.6% or greater,99.7% or greater, preferably 99.8% or greater, and more preferably 99.9%or greater, more preferably 99.95% or greater, even more preferably99.99% or greater. In some embodiments, the chiral purity of the finalpramipexole salt may be 100%.

Chirally pure pramipexole also may be prepared by the process oftrituration of a single enantiomer of pramipexole from a mixture of R(+)and (S)-pramipexole by acid addition, based on insolubility of theenantiomers in the resulting achiral salt solution. The processcomprises dissolving an enantiomerically enriched pramipexole in anorganic solvent at an elevated temperature, adding from about 1.05 molarequivalents to about 2.05 molar equivalents of a selected acid, coolingthe reaction to room temperature, stirring the cooled reaction at roomtemperature for an extended time and recovering enantiomerically purepramipexole.

In embodiments, the elevated temperature of the reaction may be belowthe boiling temperature of the reaction, specifically, below the boilingtemperature of the organic solvent(s) of the reaction mixture. Theelevated temperature may be lower than about 125° C., more preferablylower than about 100° C., and more preferably about 75° C. The timesnecessary for the reaction may vary with the identities of thereactants, the solvent system and with the chosen temperature, and wouldbe appreciated by one of skill in the art.

In embodiments, the organic solvent may include, but is not limited to,acetonitrile, acetone, ethanol, ethyl acetate, methyl tert-butyl ether,methyl ethyl ketone, isopropyl acetate and isopropyl alcohol. In apreferred embodiment, the organic solvent is ethanol. In thisembodiment, the acid may include, but is not limited to, halogenic acidssuch as, for example, hydrobromic, hydrochloric, hydrofluoric andhydroiodic acid; inorganic acids such as, for example, nitric,perchloric, sulfuric and phosphoric acid; organic acids such as, forexample, sulfonic acids (methanesulfonic, trifluoromethan sulfonic,ethanesulfonic, benzenesulfonic or p-toluenesulfonic), acetic, malic,fumaric, succinic, citric, benzoic, gluconic, lactic, mandelic, mucic,pamoic, pantothenic, oxalic and maleic acid; and aminoacids such asaspartic or glutamic acid. The acid may be a mono- or di-acid, such as adi-hydrohalogenic, di-sulfuric, di-phosphoric or di-organic acid. In allcases, the acid is used as an achiral reagent which is not selected onthe basis of any expected or known preference for interaction with orprecipitation of a specific optical isomer of the products of thisdisclosure. In a preferred embodiment, the selected acid isp-toluenesulfonic acid.

In additional embodiments, after the enantiomerically enrichedpramipexole is dissolved in an organic solvent at an elevatedtemperature and the acid is added, the reaction may be cooled to roomtemperature at a rate of about 25° C./hour. The chirally purepramipexole may then be recovered from the reaction solution by stirringthe reaction for at least about 2 hours, filtering the reaction toisolate a solid precipitate, washing the precipitate with an alcohol anddrying the precipitate under vacuum. The rates of cooling and the timerequired for the additional stirring may vary with the chosen organicsolvent and acid, and would be appreciated by one skilled in the art.Additionally, the reaction volumes may dictate the degree of opticalpurification and the overall yield of the final pramipexole product.These volumes would be understood and appreciated by one of skill in theart. Examples of specific times, temperatures and volumes which enablethe practice of this invention are given in the Examples.

In embodiments, the chiral purity for the R(+) enantiomer of therecovered pramipexole salt may be greater than 99% when the startingpramipexole material has a chiral purity for the R(+) enantiomer ofgreater than 55%, preferably greater than 70%, or more preferablygreater than 90%. The chiral purity of the final pramipexole salt forthe R(+) enantiomer may be 99.6% or greater, 99.7% or greater,preferably 99.8% or greater, and more preferably 99.9% or greater, morepreferably 99.95% or greater, even more preferably 99.99% or greater. Ina most preferred embodiment, the chiral purity of the final pramipexolesalt for the R(+) enantiomer may be 100%.

The process may include dissolving an enantiomerically enrichedpramipexole in an organic solvent at an elevated temperature, addingfrom about 1.05 equivalents to about 2.05 equivalents of a selectedacid, cooling the reaction to room temperature, stirring the cooledreaction at room temperature for an extended period of time andrecovering enantiomerically pure pramipexole of formula I.

In an embodiment of the process, the selected acid is p-toluenesulfonicacid (p-TSA) and the organic solvent is ethanol. In another embodimentof the process, the elevated temperature may be from about 65° C. toabout 85° C. and the cooling occurs at a rate of about 25° C. per hour.The elevated temperature may also be a temperature lower than 125° C.,preferably lower than 100° C., and more preferably about 75° C. Thetimes necessary for the reaction may vary with the identities of thereactants, the solvent system and with the chosen temperature, and maybe easily appreciated by one of skill in the art. In yet anotherembodiment of the process, recovering enantiomerically pure pramipexolecomprises cooling the reaction to a temperature of about 25° C. andstirring the reaction for at least about 2 hours. The recovery mayfurther comprise filtering the reaction to isolate a solid precipitate,washing the precipitate with an alcohol and drying the precipitate undervacuum.

In various embodiments of the process, the organic solvent may include,but is not limited to, acetonitrile, acetone, ethanol, ethyl acetate,methyl tert-butyl ether, methyl ethyl ketone, isopropyl acetate andisopropyl alcohol. In a preferred embodiment, the organic solvent isethanol. The acid may include, but is not limited to, halogenic acidssuch as, for example, hydrobromic, hydrochloric, hydrofluoric andhydroiodic acid; inorganic acids such as, for example, nitric,perchloric, sulfuric and phosphoric acid; organic acids such as, forexample, sulfonic acids (methanesulfonic, trifluoromethan sulfonic,ethanesulfonic, benzenesulfonic or p-toluenesulfonic), acetic, malic,fumaric, succinic, citric, benzoic, gluconic, lactic, mandelic, mucic,pamoic, pantothenic, oxalic and maleic acid; and aminoacids such asaspartic or glutamic acid. The acid may be a mono- or di-acid, such as adi-hydrohalogenic, di-sulfuric, di-phosphoric or di-organic acid. In allcases, the acid is used as an achiral reagent which is not selected onthe basis of any expected or known preference for interaction with orprecipitation of a specific optical isomer of the products of thisdisclosure. In a preferred embodiment, the selected acid isp-toluenesulfonic acid.

In embodiments of the process, the final chiral purity for an R(+)enantiomer of the pramipexole salt may be greater than 99% when thestarting mixture contains pramipexole which is at least 55% opticallypure for the R(+) enantiomer, preferably 80% optically pure for the R(+)enantiomer, preferably 85% optically pure for the R(+) enantiomer, morepreferably 90% optically pure for the R(+) enantiomer and mostpreferably 95% optically pure for the R(+) enantiomer. The final chiralpurity for an S(−) enantiomer of the pramipexole salt may be greaterthan 99% when the starting mixture contains pramipexole which is atleast 55% optically pure for the S(−) enantiomer, preferably 80%optically pure for the S(−) enantiomer, preferably 85% optically purefor the S(−) enantiomer, more preferably 90% optically pure for the S(−)enantiomer and most preferably 95% optically pure for the S(−)enantiomer. The chiral purity of the final pramipexole salt maypreferably be 99.6% or greater, 99.7% or greater, preferably 99.8% orgreater, and more preferably 99.9% or greater. In a more preferredembodiment, the chiral purity of the final pramipexole salt may be 100%.

Results of example purifications using each of the several conditionswhich are embodiments of the present disclosure are listed in Table 8.

TABLE 8 Experiments for preparation of the R(+) enantiomer ofpramipexole Acid Solvent Batch Size Results p-TSA ethanol 298.7 mg Yield= 489.5 mg (90.3%) Start Chiral Purity = 91% AUC R(+) by HPLC FinalChiral Purity = 100% AUC by HPLC MSA aceto- 300.0 mg Yield = 431.8 mg(98.9%) nitrile Start Chiral Purity = 91% AUC R(+) by HPLC Final ChiralPurity = 99.23% AUC by HPLC fumaric aceto- 301.0 mg Yield = 532 mg(84.2%) (hot nitrile Start Chiral Purity = 91% AUC R(+) ethanol) by HPLCFinal Chiral Purity = 99.26% AUC by HPLC phos- aceto- 299.4 mg Yield =592 mg (~100%) phoric nitrile Start Chiral Purity = 91% AUC R(+) by HPLCFinal Chiral Purity = 100% AUC by HPLC

The chemical and chiral purity of the preparations of (R)-pramipexolemay be verified with at least HPLC, ¹³C-NMR, ¹H-NMR and FTIR. Inpreferred embodiments, the (R)-pramipexole may be synthesized by themethod described above, which yields enantiomerically pure material.Alternatively, the (R)-pramipexole may be purified from mixtures of R(+)and (S)-pramipexole using a purification scheme which is disclosed incopending U.S. Provisional Application No. 60/894,829 entitled “Methodsof Synthesizing and Purifying R(+) and (S)-pramipexole”, filed on Mar.14, 2007, and U.S. Provisional Application No. 60/894,814 entitled“Methods of Enantiomerically Purifying Chiral Compounds”, filed on Mar.14, 2007, which are incorporated herein by reference in theirentireties.

By way of explanation, and not wishing to be bound by theory, thesolubility of (R)-pramipexole and (S)-pramipexole may be the same in thetrituration step of the synthesis and purification processes. Asexample, if a synthesis process is carried out with 90 grams of the R(+)diamine and 10 grams of the S(−)diamine, and the solubility of the finalpramipexole product is 10 grams for either enantiomer, then 80 grams ofthe (R)-pramipexole product and 0 grams of the (S)-pramipexole productwould precipitate (assuming a 100% chemical conversion from the diamineand no change in molecular weight in going to the pramipexole product).That is, 10 grams of each enantiomer of pramipexole may be expected togo into solution. This would lead to a pramipexole product with a 100%chiral purity for the R(+) enantiomer. The opposite ratio of startingmaterials for the synthesis process (90 grams of the S(−) diamine and 10grams of the R(+) diamine) may generate a reaction product of 90 gramsof the (S)-pramipexole and 10 grams of the (R)-pramipexole. From thisreaction product mixture, 80 grams of the S(−) enantiomer and 0 grams ofthe R(+) enantiomer of pramipexole would be expected to precipitate,leading to a pramipexole product with a 100% chiral purity for the S(−)enantiomer. In this thought experiment, one can imagine that the volumeswhich are used for a reaction may have a large potential effect on thefinal yield and chiral purity. That is, too large a volume will reducethe yield as more of the pramipexole enantiomer products will go intosolution (but increase the chiral purity) and too small a volume willincrease the yield as less of the pramipexole products will go intosolution (but reduce the chiral purity).

To better define the actual limits of the reaction volumes and opticalpurities attainable using methods of the disclosure, various ratios ofchiral purity for the starting diamine material were tested. As shown inTable 9, the synthesis and purification process was tested using thefollowing ratios of the starting R(+) and S(−) diamine: 80:20, 20:80,85:15, 15:85, 90:10, 10:90, 95:5 and 5:95. Additionally, three specificreaction conditions were tested which varied either the reaction volumeor a post reaction recovery step. These trials demonstrated that theenantiomers of pramipexole are equally insoluble (or soluble) in theorganic solvents utilized in the various embodiments of the synthesisprocesses disclosed herein.

TABLE 9 Experiments for S_(N)2 preparation of pure enantiomers ofpramipexole Ratio of starting Condition C Condition D Condition Ediamines (yield/chiral (yield/chiral (yield/chiral R(+):S(−) purity)purity) purity) 80:20 — 29%/99%  34%/98.2% 20:80 — 30%/99.4% 35%/95.7%85:15 43%/86.8% 36%/99.8% 39%/99.9% 15:85 52%/88.9% 27%/99.6% 37%/99.9%90:10 47%/95.9% — — 10:90 58%/93.6% — — 95:5  50%/99.6% — —  5:9547%/99.6% — — Condition C: The reaction is performed in 10 volumes ofDMF and 1.25 equivalents of propyl tosylate at 65-67° C. The reaction isthen cooled to room temperature and diluted with 8 volumes of MTBE.Condition D: The reaction is performed in 18 volumes of DMF and 1.25equivalents of propyl tosylate at 65-67° C. The reaction is then cooledto room temperature and diluted with 8 volumes of MTBE. Condition E: Thereaction is performed in 10 volumes of DMF and 1.25 equivalents ofpropyl tosylate at 65-67° C. The reaction is then cooled to roomtemperature with no dilution in MTBE.

The data in Table 9 demonstrate that both enantiomers of pramipexolehave similar, if not the same, solubility. Further, the data show thatthe synthesis is equally efficient for either enantiomer of pramipexole.These data also demonstrate that the enantiomers behave independently ofone another, in that the solubility of one enantiomer appears to beunaffected by the concentration in solution of the other. For example,the various synthesis reactions carried out using condition C all havechemical yields of about 50%, independent of the percentage ofpredominant diamine enantiomer of the starting material. When the volumeof the organic solvent used in the synthesis reaction is increased, thechemical yield is reduced, but the chiral yield is increased. This isapparent by comparison of the reaction carried out in conditions C andD, where an 85:15 ratio of R(+):S(−) diamine produced a pramipexoleproduct having an 86.8% chiral purity for the R(+) enantiomer when thereaction used 10 volumes of the organic solvent and a 99.8% chiralpurity for the R(+) enantiomer when the reaction used 18 volumes of theorganic solvent. Note also that the chemical yield was reduced in thereaction using a larger volume of organic solvent (43% yield incondition C and 36% yield in condition D).

In Table 9, condition E is the same as condition C, except that therecovery step does not incorporate dilution in MTBE. The MTBE isobserved to increase pramipexole recovery (yield) from the synthesisreaction, but may reduce the overall chiral purity. This is born out bya comparison of the results for trials carried out in an 85:15 ratio ofR(+):S(−) diamine, which produced a pramipexole product having a 86.8%chiral purity for the R(+) enantiomer when the reaction included theMTBE organic solvent and a 99.9% chiral purity for the R(+) enantiomerwhen the reaction did not include the MTBE organic solvent. The chemicalyield was reduced by exclusion of the MTBE dilution in the recoverystep; a 43% yield in condition C as opposed to a 39% yield in conditionE.

The chirally pure (R)-pramipexole prepared by any of the above methodsmay be converted to a pharmaceutically acceptable salt of(R)-pramipexole. For example, (R)-pramipexole dihydrochloride is apreferred pharmaceutical salt due its high water solubility.(R)-pramipexole dihydrochloride may be prepared from other salts of(R)-pramipexole in a one step method comprising reacting the(R)-pramipexole, or (R)-pramipexole salt, with concentrated HCl in anorganic solvent, such as an alcohol, at a reduced temperature. In someembodiments, the reduced temperature is a temperature of from about 0°C. to about 5° C. An organic solvent, such as methyl tert-butyl ether,may be added, and the reaction may be stirred for an additional hour.The (R)-pramipexole dihydrochloride product may be recovered from thereaction mixture by filtering, washing with an alcohol and vacuumdrying.

Each of the methods disclosed herein for the manufacture andpurification of (R)-pramipexole or a pharmaceutically acceptable saltthereof may be scalable to provide industrial scale quantities andyields, supplying products with both high chemical and chiral purity. Insome embodiments, the enantiomerically pure (R)-pramipexole may bemanufactured in large batch quantities as may be required to meet theneeds of a large scale pharmaceutical use.

Various aspects of the present invention will be illustrated withreference to the following non-limiting examples.

EXAMPLES Example 1—Measurement of the Dopamine Receptor Affinities forthe R(+) and S(−) Enantiomers of Pramipexole

The S(−) enantiomer of pramipexole has historically been characterizedas a high affinity dopamine receptor ligand at the D₂ (both the S and Lisoforms), D₃ and D₄ receptors, although the highest affinity is seenfor the D₃ receptor subtype. The dopamine receptor ligand affinity of(S)-pramipexole and of (R)-pramipexole from journal publications hasbeen tabulated (data are reproduced in Table 10). Although theconditions under which each study or experiment was carried out areslightly different, and different radio-ligands were used, the data showcomparable affinities for the various dopamine receptors. Studies weconducted on the dopamine receptor affinities of the S(−) and the R(+)enantiomers of pramipexole are also shown in Table 10. These datademonstrate an unexpectedly large difference in the affinities of thetwo enantiomers of pramipexole for all dopamine receptors. Table 10shows that; instead of the expected 10-20 fold difference in bindingaffinity for D2 receptor affinity, and 50-fold difference in bindingaffinity for D3 receptor affinity as derived from the literature, thevalues we found were typically 10-fold higher (290- and 649-fold,respectively) (Table 10).

TABLE 10 Comparative binding affinity data for pramipexole enantiomersReceptor Source S(−) R(+) Ratio S:R D₂ binding, IC₅₀ (nM) Lit. * 4,70043,000 9 D₂ binding, IC₅₀ (nM) Lit. ** 402 8,330 21 D₂ binding, IC₅₀(nM) Actual*** 6.2 1,800 290 D₃ binding, IC₅₀ (nM) Lit. ** 4.2 211 50 D₃binding, IC₅₀ (nM) Actual*** 0.94 610 649 * Schneider, C. S.; Mierau,J., “Dopamine Autoreceptor Agonists: Resolution and PharmacologicalActivity of 2,6-Diaminotetrahydrobenzothiazole and an AminothiazoleAnalogue of Apomorohine”, (1987). J. Med. Chem. 30: 494-498 ** Wong, S.K.-F.; Shrikhande, A. V., S. K.-F. Wong, “Activation of ExtracellularSignal-Regulated Kinase by Dopamine D2 and D3 Receptors”,. (2003)Society for Neuroscience Abstracts ***Data from current study

The (R)-pramipexole and (S)-pramipexole were supplied as dry powder toour contract research partner Cerep by the manufacturer AMRI. Solutionsof (R)-pramipexole and (S)-pramipexole were prepared from stocksolutions in DMSO. Eight concentrations of (R)-pramipexole or(S)-pramipexole (0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 mM, 10 mM and100 mM) were used to displace standard reference radiolabeled dopamineagonists. These concentrations were tested in cell lines expressingselect human cloned dopamine receptors (D_(2S), D₃). Previous work inthe literature and our data demonstrated no significant interaction withD1 and D5 dopamine receptors. Group results for the interaction of(R)-pramipexole or (S)-pramipexole with each receptor are expressed asthe IC50 in Table 10.

These data indicate that IC₅₀ values of (R)-pramipexole at thesereceptors are approximately 290 to 649 times that of the IC₅₀ values for(S)-pramipexole. Further, these data suggest that the ratio of the IC₅₀values for (R)-pramipexole to (S)-pramipexole at the D₂ receptor areapproximately 14 to 32 times larger than the ratios suggested by theliterature, at least when the chiral purities were beyond the limits ofdetection (LOD 0.05%) (chiral purity greater than 99.95%). Similarly,the data suggest that the ratio of the IC₅₀ values for (R)-pramipexoleto (S)-pramipexole at the D₃ receptor are approximately 13 times largerthan the ratios suggested by the literature, at least when the chiralpurities were beyond the limits of detection (chiral purity greater than99.95%). These data also suggest that if dopamine receptor affinity isthe major contributing factor to limiting dose tolerance of the S(−)enantiomer, then pure preparations of the R(+) enantiomer should have amaximum tolerated dose (MTD) and/or a no observable adverse effect leveldose (NOAEL) of at least 290 times greater than the S(−) enantiomer'sMTD and/or NOAEL. Thus, even a small contamination of the(R)-pramipexole compositions of the present invention by the S(−)enantiomer, at levels as low as 0.5% or less, may effect the observedMTD and NOEL.

Example 2

In vivo studies to determine the MTD and NOAEL in dogs for 100% purepreparations of the R(+) and S(−) enantiomers of pramipexole, and amixture (R 99.5%/S 0.5%). The form of (R)-pramipexole was(R)-pramipexole dihydrochloride monohydrate.

The following in vivo study in beagle dogs was undertaken to test thehypothesis that the large observed difference in receptor bindingaffinities for the R(+) and S(−) enantiomers of pramipexole willtranslate to a large observed difference in the observed maximumtolerated dose (MTD) and/or no observable adverse effect level (NOAEL)of the two enantiomers. Dogs were administered preparations of eachenantiomer prepared as a highly purified compound (100% purepreparations (within the limits of analytical detectability)), or apreparation of the R(+) enantiomer contaminated by 0.5% of the S(−)enantiomer of pramipexole.

Three groups of four non-naïve male beagle dogs were used in the study.Each group was administered various doses of either the R(+) or S(−)enantiomer prepared as a highly purified compound, or a preparation ofthe R(+) enantiomer contaminated by 0.5% of the S(−) enantiomer ofpramipexole. Doses were administered orally by gavage and clinicalobservations were taken continuously following dosing: hourly for thefirst four hours, and then twice daily cage-side observations for theduration of the inter-dose or post-dose interval. Observations were madeof clinical signs, mortality, injury and availability of food and water.Animals were fasted for 24 hr prior to dosing. Dogs in each group wereexposed to only one drug, or the combination; each dose was administeredonly once, with a subsequent dose administered after a recovery periodof 4 days. The data are summarized in Table 11.

A NOAEL was established at a dose level of 25 mg/kg for the R(+)enantiomer when administered to non-naïve dogs, while a dose level of 75mg/kg may be considered an MTD in non-naïve dogs. For the S(−)enantiomer, a NOAEL of 0.00125 mg/kg and an MTD of 0.0075 mg/kg wasfound in non-naïve dogs. For the composition containing a mixture of thetwo enantiomers (99.5% (R)-pramipexole and 0.5% (S)-pramipexole), theNOAEL was found to be 0.25 mg/kg, which corresponds to a dose of 00125mg/kg of the S(−) enantiomer, while the MTD is 1.5 mg/kg, whichcorresponds to a dose of 0.0075 mg/kg of the S(−) enantiomer. These dataindicate that the NOAEL for the R(+) enantiomer of pramipexole isapproximately 20,000-fold greater than for the S(−) enantiomer innon-naïve dogs, while the MTD is about 10,000-fold greater.

TABLE 11 Clinical observations in male beagle dogs for administration ofpramipexole compositions SUMMARY OF CLINICAL FINDINGS* Dose Amount(mg/kg) 7.5 25 75 0.0075 0.025 0.00125 1.5 5 0.25 R(+) R(+) R(+) S(−)S(−) S(−) mixture** mixture mixture (Day (Day (Day (Day (Day Day (Day(Day (Day 1) 4) 8) 1) 4) 8) 1) 4) 8) Behavior/Activity Activitydecreased 0/4 0/4 2/4 3/4 4/4 0/4 4/4 4/4 0/4 Convulsions-clonic 0/4 0/41/4 0/4 0/4 0/4 0/4 0/4 0/4 Salivation 0/4 0/4 3/4 0/4 0/4 0/4 0/4 0/40/4 Tremors 0/4 0/4 4/4 1/4 3/4 0/4 1/4 2/4 0/4 Excretion Emesis 0/4 0/42/4 3/4 4/4 0/4 1/4 3/4 1/4 Feces hard 1/4 0/4 0/4 1/4 0/4 0/4 0/4 0/40/4 Feces mucoid 0/4 0/4 0/4 0/4 0/4 0/4 1/4 1/4 0/4 Feces soft 0/4 0/41/4 0/4 0/4 0/4 2/4 1/4 1/4 Feces watery 0/4 0/4 0/4 0/4 0/4 0/4 1/4 1/40/4 External Appearance Lacrimation 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4Eye/Ocular Pupils dilated 0/4 0/4 2/4 0/4 0/4 0/4 0/4 0/4 0/4Pelage/Skin Skin warm to touch 1/4 0/4 1/4 0/4 0/4 0/4 0/4 0/4 0/4*Number of animals affected/Total number of animals **Mixture of 99.5%(R)-pramipexole and 0.5% (S)-pramipexole.

The data shown in Table 11 indicate that the receptor affinitiesidentified (see Table 10) contribute in a straightforward fashion to theobserved differences in the MTD and NOAEL doses for the R(+) and S(−)enantiomers of pramipexole. These data also indicate that the chiralpurity for the R(+) enantiomer of pramipexole in embodiments of thecompositions of the present invention (refer to Tables 5 and 6) may needto be in excess of 99.9%, depending on the final total dose, to avoidthe adverse side effects of (S)-pramipexole.

Further, the data in Table 11 demonstrate that the NOAEL and MTD for thecombination composition (99.5% (R)-pramipexole and 0.5% (S)-pramipexole)may be determined directly by the dose of the S(−) enantiomer in thecomposition. Thus, a small (fractional percentage) contamination of acomposition of (R)-pramipexole by the S(−) enantiomer may reduce the MTDand NOEL of the composition. For example, in these experiments, the MTDof pramipexole was reduced from 75 mg/kg for the R(+) enantiomer to atotal dose of 1.5 mg/kg of the mixed composition (a factor of 50), andthe NOAEL was reduced from 25 mg/kg to 0.25 mg/kg, respectively (afactor of 100). Since the shift in MTD and NOAEL may be predicted by thedose of the S(−) enantiomer of pramipexole in the mixture, the shift forany unknown mixture may be calculated based on the percentagecontamination of the (R)-pramipexole by the S(−) enantiomer, relative tothe MTD and NOAEL for (S)-pramipexole. This indicates that anycontamination of an (R)-pramipexole dosing solution with (S)-pramipexolewill have a measurable effect on these indicators of dose tolerability.

Example 3.1-Toxicology Studies in Rats and Minipigs and Phase I Studiesin Healthy Adult Volunteers

Two-week and three-month toxicology studies of (R)-pramipexole in ratsand minipigs were completed. NOAEL dose levels of 150 mg/kg at two-weeksand 100 mg/kg at three-months for rats and 75 mg/kg at two-weeks and 50mg/kg at three-months for minipigs were established. Phase I studies ofhealthy adult volunteers have demonstrated that (R)-pramipexole inascending single doses up to 300 mg and multiple doses up to 200 mg perday for 4½ days is safe and well-tolerated. The Mirapex® label specifiesa starting dose of 0.125 mg and a maximum total daily dose of 4.5 mg.The Phase I data demonstrate, therefore, that (R)-pramipexole may besafely administered (1) at starting doses that are at least 2400-foldhigher than the Mirapex® starting dose and (2) at steady state dosesthat are at least 44-fold higher than the highest recommended dose ofMirapex®. The form of (R)-pramipexole was (R)-pramipexoledihydrochloride monohydrate.

The preliminary results of the clinical studies and the toxicologystudies are discussed. Exposure at steady state in rats, minipigs, andhumans is linear across all doses studied. After 3 months of dosing, thecurrent No Observed Adverse Effect Level (NOAEL) in rats has beendetermined to be 100 mg/kg; and the current NOAEL in minipigs has beendetermined to be 50 mg/kg. The mean steady state AUC in rats at theNOAEL dose of 100 mg/kg was 61,299 and 61,484 h*ng/mL for males andfemales, respectively, and for minipigs at the NOAEL dose of 50 mg/kgwas 91,812 and 131,731 h*ng/mL for males and females, respectively. Themean steady state AUC in humans at a dose of 100 mg Q12H (200 mg totaldaily dose) was 2,574 h*ng/mL. The drug has been safe, well-tolerated,and free of clinically significant adverse events in healthy adultsubjects at single doses up to 300 mg and at multiple doses up to 100 mgQ12H, and the projected human exposure associated with a daily dose of250 mg Q12H is expected to be greater than 13-fold lower than exposuresseen at the NOAEL in male minipigs and approximately 9-fold lower thanexposures seen at the NOAEL in male and female rats after 13 weeks ofdosing.

3.2—Clinical Studies.

(R)-pramipexole has been studied at single daily doses of 50, 150 and300 mg and twice daily doses of 50 and 100 mg for 4½ days in healthyadult volunteers. The drug has been safe and well-tolerated in bothstudies and there were no serious adverse events, discontinuations dueto adverse events, or dose-related or clinically significant adverseevents in either study. The most frequent adverse events have beendizziness and headache, all of which have been mild to moderate inseverity and resolved without intervention.

3.2.1—Summary of (Blinded) Safety and Pharmacokinetic Results of(R)-Pramipexole (Ascending Single-Dose Study).

Three sequential panels of 8 subjects each received single doses of(R)-pramipexole (6 subjects) or placebo (2 subjects) at ascending doselevels of 50, 150, and 300 mg. Safety observations included vital signs,physical examination, clinical laboratory tests, ECGs, and adverse eventreporting. Blood and urine samples were collected pre-dose and for 72hours post-dose to assess the pharmacokinetics. All 24 subjectscompleted the study as planned. There were no serious adverse events;46% of all subjects reported at least one non-serious adverse event(AE). Most AEs were mild; the most frequent AE was mild dizziness in 21%of subjects. There were no clinically significant safety observations atany dose level.

Pharmacokinetic data indicated that (R)-pramipexole is rapidly absorbedwith mean maximum concentrations of 125, 360, and 781 ng/mL reached atapproximately 2 hours post-dose for the 50, 150, and 300 mg dose groups,respectively (see FIG. 1 and Table 12, below). Mean exposures(AUC_(0-∞)) were 1254, 3815, and 8623 h*ng/mL for the 50, 150, and 300mg dose groups, respectively. Both C_(max) and AUC increased inproportion to dose across the dose levels tested. Urinary excretion ofunchanged drug accounted for approximately 70% of drug eliminationacross dose levels. The mean T_(1/2) was 6-7 hours and was independentof dose. Comparison of the mean plasma concentrations (FIG. 2) and meanpharmacokinetic parameters (Table 12) after administration of a single150 mg following a high fat/high calorie breakfast with those afteradministration of 150 mg under fasted conditions demonstratesessentially no effect of a meal on the absorption and elimination of(R)-pramipexole.

Results of this study demonstrate that single oral doses of 50, 150, and300 mg (R)-pramipexole are safe and well-tolerated. The drug is orallybioavailable and the pharmacokinetics are linear. Absorption andelimination are not affected by a high fat/high calorie meal.

TABLE 12 Summary of pharmacokinetic parameters for (R)-pramipexole afteroral administration of single 50 mg, 150 mg, and 300 mg doses to healthyvolunteers under fasted conditions and 150 mg under fed conditions.Fasted Fed Parameter¹ 50 mg 150 mg 300 mg 150 mg Cmax(ng/mL)  125 ± 22.0(6)  360 ± 60.4 (6) 781 ± 158 (6) 315 ± 062 (6) Tmax(h) 2.04 (6) 2.04(6) 1.96 (6) 2.58 (6) AUC(0-t) 989 ± 295 (6) 3,387 ± 746 (6)  8,339 ±3,202 (6) 3,099 ± 920 (6)  (h · ng/mL) AUC(inf) 1,254 ± 347 (6)  3,815 ±972 (5)  8,623 ± 3,262 (6) 3,397 ± 944 (6)  (h · ng/mL) λz 0.1064 ±0.0171 (6) 0.1001 ± 0.0087 (5) 0.1151 ± 0.0309 (6) 0.1152 ± 0.0256 (6)(h⁻¹) t½ (h) 6.65 ± 1.07 (6) 6.96 ± 0.56 (5) 6.40 ± 1.73 (6) 6.28 ± 1.48(6) CL/F (mL/min) 706 ± 182 (6) 692 ± 183 (5) 659 ± 260 (6) 774 ± 165(6) Vz/F (L)  395 ± 61.9 (6) 411 ± 081 (5)  346 ± 98.5 (6)  406 ± 62.8(6) Ue (mg) 35.3 ± 5.19 (6) 60.5 ± 7.04 (6)  198 ± 28.0 (6) . ± . (0) Fe(% Dose) 70.7 ± 10.4 (6) 40.3 ± 4.69 (6) 65.8 ± 9.33 (6) . ± . (0) CLr(mL/min) 628 ± 149 (6)  310 ± 74.3 (6) 441 ± 159 (6) . ± . (0) ¹Mean ±standard deviation (N) except for Tmax for which the median (N) isreported.

3.2.2—Summary of (Blinded) Safety and Pharmacokinetic Results of(R)-Pramipexole (Ascending Multiple-Dose Study).

This study is ongoing and has not yet been unblinded with respect totreatment assignments, and only clinical observations andpharmacokinetic data are available for the first 2 panels. To date, 2sequential panels of 8 subjects each were enrolled to receive multipledoses of (R)-pramipexole (6 subjects) or placebo (2 subjects). The firstpanel was administered a singe dose of 50 mg, followed 48 hours later by4½ days of multiple dosing (twice daily) at 50 mg Q12 hours. The secondpanel was administered a singe dose of 100 mg, followed 48 hours laterby 4½ days of multiple dosing (twice daily) at 100 mg Q12 hours. Safetyobservations included vital signs, physical examination, clinicallaboratory tests, ECGs, and adverse event reporting. Blood samples werecollected pre-dose on Day 1 and serially for 48 hours post-dose toassess the single-dose pharmacokinetics. Blood samples were collectedpre-dose on Days 5, 6, and 7 to confirm steady-state was achieved, andserially through 72 hours post-dose on Day 7 to assess the steady-statepharmacokinetics of (R)-pramipexole. Urine samples were collected for 12hours after dosing on Day 7 to assess urinary excretion.

All 16 subjects enrolled to date have completed the study as planned.There were no deaths, reports of serious adverse events, ordiscontinuations because of adverse events during the study. Both doselevels were well tolerated. In cohort 1, all adverse events were mild inintensity, with the exception of moderate headaches reported by 2subjects. In cohort 2, all adverse events were mild in intensity, withthe exception of moderate “stiffness in back” and a moderate vasovagalresponse reported in 1 subject. An asymptomatic mild increase in heartrate upon standing (without change in blood pressure) was reported bythe principal investigator for 1 of the 8 subjects dosed in cohort 1 (50mg cohort) and for 2 of the 8 subjects dosed in cohort 2 (100 mgcohort). There were no clinically significant safety observations at anydose level.

Pharmacokinetic data are shown in Table 13 and FIG. 3. C_(m)ax andAUC₍₀₋₁₂₎ increased 37% and 40%, respectively from Day 1 to Day 7 forsubjects receiving 50 mg Q12H, with essentially no change in T_(max).Mean exposure AUC₍₀₋₁₂₎ at Day 7 was 1449 h*ng/mL for the 50 mg Q12Hdose group. C_(max) and AUC₍₀₋₁₂₎ increased 24% and 38%, respectivelyfrom Day 1 to Day 7 for subjects receiving 100 mg Q12H, with essentiallyno change in T_(max). Mean exposure AUC₍₀₋₁₂₎ at Day 7 was 2465 h*ng/mLfor the 100 mg Q12H dose group. Results of this study demonstrate thatmultiple oral doses of 50 and 100 mg (R)-pramipexole administered twicedaily are safe and well-tolerated. The drug is orally bioavailable andthe pharmacokinetics are linear at steady state, with no significantaccumulation.

TABLE 13 Summary of pharmacokinetic parameters for (R)-pramipexoleduring oral administration of 50 mg and 100 mg doses on Day 1, Q12H onDays 3 through 6, and a single dose on Day 7 to healthy volunteers underfasted conditions. Dose Parameter¹ 50 mg 100 mg Day 1 Cmax (ng/mL)  139± 15.3 (6)  248 ± 30.4 (6) Tmax (h) 1.83 (6) 1.92 (6) AUC(0-12) (h ·ng/mL) 1,035 ± 121 (6)  1,776 ± 260 (6)  AUC(0-t) (h · ng/mL) 1,463 ±280 (6)  2,545 ± 497 (6)  AUC(inf) (h · ng/mL) 1,502 ± 280 (6)  2,574 ±505 (6)  λz (h⁻¹) 0.1132 ± 0.0230 (6) 0.1073 ± 0.0161 (6) t½ (h) 6.34 ±1.31 (6) 6.57 ± 0.88 (6) CL/F (mL/Min) 571 ± 107 (6) 665 ± 107 (6) Vz/F(L)  306 ± 45.8 (6)  373 ± 51.0 (6) Day 7 Cmax (ng/mL)  191 ± 20.9 (6)306 ± 055 (6) Tmax (h) 1.75 (6) 2.00 (6) AUC(0-12) (h · ng/mL) 1,449 ±221 (6)  2,465 ± 299 (6)  λz (h⁻¹) 0.1025 ± 0.0186 (6) 0.0894 ± 0.0117(6) t½ (h) 6.96 ± 1.30 (6) 7.88 ± 1.19 (6) CL/F (mL/Min)  585 ± 81.6 (6) 684 ± 76.1 (6) Vz/F (L)  346 ± 30.1 (6)  466 ± 82.2 (6) Ue (mg) . ± .(0) . ± . (0) Fe (% Dose) . ± . (0) . ± . (0) CLr (mL/min) . ± . (0) . ±. (0) ¹Mean ± standard deviation (N) except for Tmax for which themedian (N) is reported.

3.3—Toxicology Studies.

In a 2-week repeat-dose toxicology studies in rats, animals received 50,150, and 500 mg/kg doses of (R)-pramipexole for 14 days. (R)-pramipexolecaused mortality at the high dose of 500 mg/kg and statisticallysignificant changes in body weight gain and food consumption for bothsexes were observed in the animals surviving to terminal sacrifice. Notarget organ toxicity by histopathology examination was identified atany dose. The NOAEL for this 2-week study in rats was determined to be150 mg/kg. Following this study, 3- and 6-month repeat dose toxicologystudies were completed at doses of 30, 100, and 300 mg/kg. The resultsof the 3-month study contain some target organ toxicity byhistopathology examination at the highest dose (300 mg/kg) with no testarticle related deaths and no significant clinical observations outsideof several incidences of convulsions in high dose rats lastingapproximately 2 minutes. The animals' health did not appear to beotherwise adversely affected by these convulsions. Test article-relatedmicroscopic changes were observed in the liver (minimal gradecholestasis correlating with increased total bilirubin), ileal smallintestine (minimal grade mineralization), and thymus (minimal gradelymphoid depletion correlating with lower group thymus weights comparedto controls). The NOAEL for the 3-month study in rats is considered tobe 100 mg/kg. Systemic exposure (AUC_(0-last)) at week 13 at the NOAELdose of 100 mg/kg was 61,299 h*ng/mL in males and 61,484 h*ng/mL infemales. The in-life phase of the 6-month toxicology study in rats wasrecently completed and histopatholgic examinations are pending. Therewere no mortalities at any dose level between the 13-week and 26-weeksacrifices.

In a 2-week repeat-dose toxicology study in minipigs, animals received7.5, 25 and 75 mg/kg doses of (R)-pramipexole for 14 days. No targetorgan toxicity by histopathology examination was identified at any dose.Clinical observations included salivation, decreased activity, emesisand inappetance, with higher incidences of emesis in females than males,and mostly in the 75 mg/kg group. The incidence of emesis at 75 mg/kg(at least one episode in 5 of 8 animals dosed at 75 mg/kg) suggestedthis dose is close to the limit of tolerability for (R)-pramipexole forchronic dosing in minipigs. Since no test article related toxicologicalchanges were observed at the high dose, the NOAEL for the 2-week studywas considered to be greater than or equal to 75 mg/kg. Based on thisstudy, 3-, and 6-, and 9-month repeat dose studies of (R)-pramipexole inminipigs were initiated at dose levels of 7.5, 25 and 75 mg/kg. At month2, dose levels were reduced to 7.5, 25 and 50 mg/kg due to mortalitiesat the 75 mg/kg level. The 3- and 6-month repeat dose studies have nowbeen completed at the 7.5, 25 and 50 mg/kg dose levels and the 9-monthrepeat dose study is ongoing. No target organ toxicity by histopathologyexamination was identified at any dose level following animal sacrificeafter 3 months of exposure. The NOAEL for the 3-month study in minipigsis considered to be 50 mg/kg. Systemic exposure (AUC0-24) at week 13 atthe NOAEL dose of 50 mg/kg/day was 91,812 h*ng/mL in males and 131,731h*ng/mL in females. The in-life phase of the 6-month toxicology study inminipigs was recently completed and histopatholgic examinations arepending. There were no mortalities attributed to test article orsignificant clinical observations at any dose level between the 13-weekand 26-week sacrifices. The ongoing 9-month toxicology studies inminipigs have now passed month 7 and no deaths attributed to testarticle or significant clinical observations have occurred at any doselevel.

3.4. Human Dosages.

The development of (R)-pramipexole as a treatment of ALS is based on amaximally tolerated dose strategy, derived either from tolerability orsafety data from studies in humans or from the results of animaltoxicology studies. To date there have been no dose-limitingtolerability observations in humans. Therefore, in order to progressdosing in humans, it is necessary to closely examine the exposure atwhich toxicity has been observed in rats and minipigs. Pharmacokineticdata obtained to date suggest that pharmacokinetics in humans willcontinue to be dose-proportional at higher doses, and that theaccumulation factor will be constant. Safety and toxicokinetic resultsfrom the 3 month toxicology studies in rats and minipigs show no adverseeffects of chronic dosing up to 100 mg/kg in rats and 50 mg/kg inminipigs. Analysis of safety margins in (R)-pramipexole exposure betweenthe NOAEL for minipigs and the projections of human exposure, therefore,support progression of total daily doses up to 500 mg in humans. Theprojected steady-state exposure of (R)-pramipexole at a total daily doseof 500 mg administered as 250 mg Q12H is approximately 7,000 h*ng/mL,which is greater than 13-fold lower than exposures seen at the NOAEL inmale minipigs and approximately 9-fold lower than exposures seen at theNOAEL in male and female rats after 13 weeks of dosing.

FIGS. 4 and 5 are plots of exposure vs. dose for rats and minipigs,respectively, compared with humans. Each graph displays the relationshipbetween exposure as expressed by AUC (h*ng/mL) and dose as expressed bybody surface area (mg/m2) at every dose level administered to eachspecies in both the 2-week and 13-week assessments. Individual datapoints with error bars are the mean±SD. The dashed horizontal line atthe bottom of both charts illustrates the extrapolated steady state AUC(7,000 h*ng/mL) in humans at 250 mg Q12H. Table 17A and Table 17B are anintegrated summary of all human pharmacokinetic estimates obtained inthe two Phase I studies.

TABLE 17A Summary of the human pharmacokinetic estimates obtained in thetwo Phase I studies with healthy volunteers Dose Dosing Cmax TmaxAUC(0-t) AUC (inf) AUC(0-12) Study (mg) Regimen Food (ng/mL) (h)(h*ng/mL) (h*ng/mL) (h*ng/mL) CL001 50 SD Fasted 125 ± 2.04 (6)   989 ±1,245 ± 347 (6)   — 22.0 (6) 295 (6) 150 SD Fasted 360 ± 2.04 (6) 3,387± 3,815 ± 972 (5)   — 60.4 (6) 746 (6) 300 SD Fasted 781 ± 1.96 (6)8,339 ± 8,623 ± 3,262 (6) — 158 (6) 3,202 (6) 150 SD Fasted 315 ± 2.58(6) 3,099 ± 3,397 ± 944 (6)   — 062 (6) 920 (6) CL002 50 Q12 H Fasted139 ± 1.83 (6) 1,463 ± 1,502 ± 280 (6)   1,035 ± 121 (6) (Day 1) 15.3(6) 280 (6) (Day 7) Fasted 191 ± 1.75 (6) — — 1,449 ± 221 (6) 20.9 (6)100 Q12 H Fasted 248 ± 1.92 (6) 2,545 ± 2,574 ± 505 (6)   1,776 ± 260(6) (Day 1) 30.4 (6) 497 (6) (Day 7) Fasted 306 ± 2.00 (6) — — 2,465 ±299 (6) 055 (6) 250 Q12 H Fasted — — — — — (Day 1) (Day 7) Fasted — — —— — Mean ± standard deviation (N) except for Tmax for which the median(N) is reported. SD = single dose

TABLE 17B Summary of the human pharmacokinetic estimates obtained in thetwo Phase I studies with healthy volunteers (continued) Dose Dosing t ½CL/F Vz/F Ue CLx Study (mg) Regimen Food (h) (mL/h) (L) (mg) (% Dose)(mL/min) L001 50 SD Fasted 6.65 ± 706 ± 395 ± 35.3 ± 5.19 (6) 70.7 =10.4 (6) 628 = 1.07 (6) 182 (6) 61.9 (6) 149 (6) 150 SD Fasted 6.96 ±692 ± 411 ± 60.5 ± 7.04 (6) 40.3 = 4.69(6) 310 = 0.56 (5) 183 (5) 081(5) 74.3 (6) 300 SD Fasted 6.40 ± 659 ± 346 ±  198 ± 28.0 (6) 65.8 =9.33(6) 441 = 1.73 (6) 260 (6) 98.5 (6) 159 (6) 150 SD Fasted 6.28 ± 774± 406 ± . ± (0) . ± (0) . ± (0) 1.48 (6) 165 (6) 62.8 (6) L002 50 Q12 HFasted 6.34 ± 571 ± 306 ± — — — (Day 1) 1.31 (6) 107 (6) 45.8 (6) (Day7) Fasted 6.96 ± 585 ± 346 ± . ± (0) . ± (0) . ± (0) 1.30 (6) 81.6 (6)30.1 (6) 100 Q12 H Fasted 6.57 ± 665 ± 373 ± — — — (Day 1) 0.88 (6) 107(6) 51.0 (6) (Day 7) Fasted 7.88 ± 684 ± 466 ± . ± (0) . ± (0) . ± (0)1.19 (6) 76.1 (6) 82.2 (6) 250 Q12 H Fasted — — — (Day 1) (Day 7) Fasted— — — Mean ± standard deviation (N) except for T_(max) for which themedian (N) is reported. SD = single dose

Exposure at steady state in rats, minipigs, and humans is linear acrossall doses studied. After 3 months of dosing, the NOAEL in rats has beendetermined to be 100 mg/kg; and the NOAEL in minipigs has beendetermined to be 50 mg/kg. The mean AUC in rats at the NOAEL was 61,299and 61,484 h*ng/mL for males and females, respectively, and for minipigswas 91,812 and 131,731 h*ng/mL for males and females, respectively. Themean AUC in humans at steady state at a dose of 100 mg Q12H (200 mgtotal daily dose) was 2,574 h*ng/mL.

Example 4—Preparation of Capsules with (R)-Pramipexole

(R)-(+)-pramipexole dihydrochloride monohydrate is filled in hardgelatin capsules with no excipients. The capsules used for the drugproduct are #00 blue opaque gelatin capsules from Hawkins ChemicalGroup. Dose strengths of 50 and 500 mg are produced. Matching placebocapsules are filled with microcrystalline cellulose. Capsules areprepared by weighing individual empty capsules and recording the weight(W_(e)). Specified amount of active drug substance are individuallyweighed and hand-filled into a capsule bottom using a Torpac® fillingfunnel. A purity adjustment factor of 1.0638 is used to adjust for thewater weight (monohydrate) in the salt form, i.e., a 50 mg dose shouldhave a target fill of 50×1.0638=53.16 mg. Capsule tops are joined withthe filled capsule bottom. The filled capsules are then weighed, and theweight is recorded (W_(f)). The calculated weight of the drug substancein the capsule (W_(f)−W_(e)) is recorded. If this calculated weight iswithin +/−5% of the nominal weight, then the capsule is cleaned,polished, and placed into and appropriately labeled container. If thecalculated weight is outside of the specified range, the capsule isdiscarded. The free-base weight per capsule (free-base weight per mg ofcapsule contents multiplied by fill weight) is 90% to 100% of thecalculated label claim. Total impuritie are ≤2%. The appearance is ablue capsule containing white to off-white powder.

Example 4B—Preparation of Tablets with (R)-Pramipexole

Capsules

with 125 mg dose strength are prepared with the composition shown inTable 17. Capsules are generally prepared under conditions of 60 to 74°F. and a relative humity of 30 to 60%. Microcrystalline cellulose,mannitol, crospovidone, magnesium stearate, and (R)-pramipexole (milled)are weighed out in the amounts shown in the column “Quantity/batch” inTable 14. The microcrystalline cellulose, mannitol, crospovidone, and(R)-pramipexole are then hand screened through a #20 mesh stainlesssteel screen and transferred to a Maxiblend V-blender with a 4 quartshell. The materials are then mixed using the Maxiblend V-blender for 10minutes. The magnesium stearate is then screened using a 30 meshstainless steel hand screen and transferred to the blender. The powdersare then mixed for five minutes. The final blend is then emptied into alabeled, double PE-lined drum and the gross, tare, and net weights arerecorded.

Tablets are prepared using a Minipress II B with 5 stations of ⅜″ round,standard, concave tooling and gravity feed frame. The final blend isplaced in the hopper and the tablet press set up is run according to thespecifications in Table 15.

TABLE 14 Tablet and Batch Compositions Quantity/ Quantity/ IngredientPercent unit (mg) batch (g) (R)-Pramipexole (milled) 40.00 125.00400.000 Microcrystalline cellulose (Avicel 35.25 110.16 352.512 PH102)(Diluent) Mannitol (Pearlitol SD100) (Diluent) 20.00 62.50 200.000Crospovidone (Polyplasdone XL) 4.00 12.50 40.000 (Disintegrant)Magnesium stearate (vegetable source, 0.75 2.34 7.488 grade 905-G)(Lubricant) Total 100.00 312.50 1000.000

TABLE 15 Tablet Press Settings Parameter Target (range) Average tabletweight (10 tablets) 3.125 g (3.031 g to 3.219 g) (+/−3%) Target weight(individual tablet) 312.5 mg (296.9 mg to 328.1 mg) (+/−5%) Targethardness 12 Kp (6 Kp to 18 Kp) Press speed 20 rpm (10 to 30 rpm)

Example 5—Preparation of (R)-Pramipexole p-TSA Salt: Condition A

All reagents were purchased from CNH technologies, Fisher, Aldrich, G.J. Chemicals, Puritan, TCI and Spectrum and were used as provided.Proton nuclear magnetic resonance spectra were obtained on a Bruker AC300 spectrometer at 300 MHz. HPLC analysis for chiral purity wasperformed on a Chiralpak® IA column (5 μM, 250×4.6 mm) at 30° C. using amobile phase of heptane/ethanol/diethylamine (80:20:2 v/v/v). HPLCanalysis for chemical purity was performed on a Sunfire® column (3.5 μM,150×4.6 mm) at 30° C. using two mobile phases: A—0.5% TFA in water; andB—0.5% TFA in methanol. A gradient of 5% B to 80% B was used to separatethe diamine and pramipexole peaks. A detection wavelength of 265 nm wasused for both HPLC analyses.

Each of the processes detailed in examples 5-14 may also be scaled forindustrial manufacturing processes, as shown in examples 15-17. Certainexamples have been detailed at both the laboratory scale and theindustrial manufacturing scale to demonstrate that the chemical andchiral yields are independent of the scale of the synthesis.

A 2.0 liter, three-necked flask was equipped with an overhead stirrer, atemperature probe, a heating mantle, a claisen joint, a refluxcondenser, and a 500 ml addition funnel. The flask was charged with 45grams of R(+)-2,6 diamino-4,5,6,7-tetrahydro-benzothiazole, followed by750 ml of n-propanol. Under continuous stirring, the mixture was heatedto a temperature of 95° C. over 15 minutes generating a clear solution.The addition funnel was charged with a solution of 74 grams propyltosylate and 60 ml diisopropylethyleamine in 250 ml n-propanol. Thissolution was added dropwise to the 2.0 liter flask with continuousstirring over a period of 4 hours. The reaction was continued withstirring for an additional 8 hours at 95° C., after which the solutionwas brought to room temperature, and stirring was continued for anadditional 4 hours.

The precipitated material was collected by filtration and washed threetimes using 100 ml reagent grade alcohol each time. The alcohol washedprecipitated cake was then washed with 100 ml heptane and dried underhigh vacuum for 2 hours. The final weight of the dried product was 53.2grams, representing a 52.2% yield. HPLC was used to determine thechemical purity of the R(+)-2,6-diamino-4,5,6,7-tetrahydro-benzothiazole((R)-pramipexole) as 98.2% and the chiral purity as greater than 99.5%.¹H NMR and ¹³C NMR were used to confirm the structure.

Example 6—Preparation of Racemic Pramipexole p-TSA Salt: Condition A

A 250 ml, three necked flask was equipped with a magnetic stirrer, atemperature probe, a heating mantle, a claisen joint, a refluxcondenser, and a 100 ml addition funnel. The flask was charged with 5grams of racemic 2,6 diamino-4,5,6,7-tetrahydro-benzothiazole, followedby 80 ml of n-propanol. Under continuous stirring, the mixture washeated to a temperature of 95° C. over 15 minutes generating a clearsolution. The addition funnel was charged with a solution of 10.12 gramspropyl tosylate and 8.2 ml diisopropylethyleamine in 28 ml n-propanol.This solution was added dropwise to the 250 ml flask with continuousstirring over a period of 2 hours. The reaction was continued withstirring for an additional 6 hours at 95° C., after which the solutionwas brought to room temperature, and stirring was continued for anadditional 6 hours.

The precipitated material was collected by filtration and washed twotimes using 25 ml reagent grade alcohol each time. The alcohol washedprecipitated cake was then washed with 25 ml heptane and dried underhigh vacuum for 1 hours. The final weight of the dried product was 5.12grams, representing a 45% yield. HPLC was used to determine the chemicalpurity of the racemic 2,6-diamino-4,5,6,7-tetrahydro-benzothiazole(racemic pramipexole) as 97.12%, and the chiral purity showed a 1:1mixture of the R(+) and (S)-pramipexole. ¹H NMR was used to confirm thestructure.

Example 7—Preparation of (R)-Pramipexole p-TSA Salt: Condition C

A 12 L, three necked flask was equipped with an overhead stirrer, atemperature probe, a heating mantle, a claisen joint, a condenser, and a500 ml addition funnel. The flask was charged with 250 grams of R(+)-2,6diamino-4,5,6,7-tetrahydro-benzothiazole (R(+) diamine), followed by 2 Lof dimethyl formamide (DMF). Under continuous stirring, the mixture washeated to a temperature of 65° C. The addition funnel was charged with asolution of 386.6 grams propyl tosylate (1.25 molar equivalents) and 322ml diisopropylethyleamine (1.25 molar equivalents) in 500 ml DMF. Thissolution was added to the 12 L flask dropwise over a period of 2.0hours. The reaction was monitored by analysis on HPLC.

The reaction was continued at 65° C. for an additional 5 hours, afterwhich the solution was gradually cooled to room temperature and stirredovernight. The solution was diluted with 2 L MTBE and stirred for anadditional 0.5 hours. The precipitated material was collected byfiltration and washed with 500 ml MTBE, followed by 3 washes of 500 mleach reagent alcohol. The washed precipitated cake was dried under highvacuum.

The final weight of the dried product was 317.6 grams, representing a56% yield. HPLC was used to determine the chemical purity of theR(+)-2,6-diamino-4,5,6,7-tetrahydro-benzothiazole ((S)-pramipexole) as98.4% and the chiral purity as greater than 99.8%. ¹H NMR and ¹³C NMRwas used to confirm the structure: 1H NMR (300 MHz, DMSO-d6) δ 8.5(br.s, 2H), 7.5 (d, 2H), 71.2 (d, 1H), 6.8 (s, 2H), 3.4 (m, 1H), 2.95(m, 3H), 2.6 (m, 2H, merged with DMSO peak), 2.3 (s, 3H), 2.15 (m, 1H),1.8 (m, 1H), 1.55 (m, 2H), 0.9 (t, 3H); ¹³C NMR (300 MHz, DMSO-d6) δ167.0, 145.5, 144.6, 138.4, 128.6, 125.8, 110.7, 53.9, 46.5, 25.8, 25.6,24.5, 21.2, 19.6, 11.3.

Example 8—Conversion of (R)-Pramipexole p-TSA Salt to (R)-Pramipexoledihydrochloride

(R)-pramipexole p-TSA salt (50 grams; 0.13 mol) was taken into 150 mlabsolute ethanol and cooled to between 0 and 5° C. with continuousstirring. Concentrated HCl (33 ml) was slowly added to the reactionwhile maintaining the temperature at between 0 and 5° C., and themixture was stirred for an additional 15 minutes. MTBE (200 ml) wasadded to the mixture, and stirring was continued for an additional 1.5hours at temperature. The reaction mixture was then filtered, washedtwice with an MTBE/ethanol solution (2:1, 2×50 ml wash volumes), anddried under vacuum at 30° C. overnight. The final product was 34 gramsof (R)-pramipexole dihydrochloride, indicative a of 92% yield, and a97.3% chemical purity as determined by HPLC.

Example 9—Conversion of (R)-Pramipexole p-TSA Salt to (R)-PramipexoleDihydrochloride

(R)-pramipexole p-TSA salt (10 grams; 0.026 mol) was dissolved in 200 mlIPAC and cooled to 15° C. with continuous stirring. HCl gas was bubbledinto the slurry for 1 hour. The mixture was then filtered, washed withIPAC, and dried overnight under vacuum at room temperature. The finalproduct was 6.8 grams of (R)-pramipexole dihydrochloride, indicative aof 92% yield, and a 97% chemical purity as determined by HPLC.

Example 10—Conversion of (R)-Pramipexole p-TSA Salt to (R)-PramipexoleFree Base

(R)-pramipexole p-TSA salt (25 grams; 0.065 mol) was dissolved in 200 mlDCM and mixed into a slurry. 10 ml of water was added and the mixturewas basified with 12 ml of 6N NaOH to a pH of 11-12. The two phases weresplit, and the aqueous was extracted with 200 ml of DCM. The combinedorganic phases were dried over MgSO₄, filtered over Celite® andconcentrated. The residue was dissolved in 100 ml MTBE and slurried forseveral hours. The solids were then filtered, washed with MTBE and driedunder vacuum at 35° C. The final product was 9.1 grams of(R)-pramipexole dihydrochloride, indicative a of 66% yield, and a 98%chemical purity as determined by HPLC.

Example 11—Conversion of (R)-Pramipexole p-TSA Salt to (R)-PramipexoleFree Base

Freebase formation was performed on a 200 gram scale. A 5 L, threenecked, round-bottomed flask, equipped with an over head stirrer,thermometer, and addition funnel was charged with 200 g (0.522 mol) of(R)-pramipexole p-TSA salt and 1 L of water. The mixture was stirred andcooled to 10° C. The slurry was basified to a pH of about 11-12 by theslow addition of 200 ml of 6 N NaOH over period of 15 min. The reactionmixture was diluted with 500 ml of brine (sodium chloride dissolved inwater) and extracted with 3×1 L of dichloromethane. The combined organicphases were washed with 1.0 L of brine, dried over MgSO₄, filtered andconcentrated to dryness. The residue was triturated with 1 L of 1:1IPAC:Heptane, the resulting slurry was stirred for 1 hour, filtered andthe filter cake was washed with 2×250 ml of 1:1 mixture of IPAC:Heptane.The filter cake was collected and dried at 40° C. under high vacuum for24 hours to give 94.1 grams (R)-pramipexole (85.5%) as a white solid.The chemical purity was 100% AUC as tested by HPLC, and the chiralpurity was 100% AUC as tested by HPLC. ¹H NMR and ¹³C NMR was used toconfirm the structure: 1H NMR (300 MHz, DMSO-δ6) δ 6.6 (s, 2H), 2.8 (m,2H), 2.5 (m, 2H, merged with DMSO peak), 2.2 (m, 1H), 1.9 (m, 1H),1.5-1.3 (m, 4H), 0.85 (t, 3H); ¹³C NMR (300 MHz, DMSO-d6) δ 166.2,144.8, 113.6, 54.2, 49.1, 30.0, 29.6, 25.2, 23.5, 12.3.

Example 12—Conversion of (R)-Pramipexole Free Base to (R)-PramipexoleDihydrochloride

The freebase of (R)-pramipexole (4.8 grams; 0.022 mol) was dissolved in200 ml of IPAC and cooled to 15° C. HCl gas was bubbled into the slurryfor 1 hour. The mixture was then filtered, washed with IPAC and driedunder vacuum at room temperature overnight. The final product was 6.4grams of (R)-pramipexole dihydrochloride, indicative a of 100% yield,and a 97% chemical purity as determined by HPLC.

Example 13—Conversion of (R)-Pramipexole Free Base to (R)-PramipexoleDihydrochloride

The freebase of (R)-pramipexole (50 grams; 0.13 mol) was dissolved in500 ml of IPAC. Under continuous stirring, the mixture was slowlycharged with 78 ml of concentrated HCl at a temperature of 25° C. Themixture was stirred overnight at ambient conditions (˜25° C.), filteredand dried under vacuum at 40° C. The final product was 68 grams of(R)-pramipexole dihydrochloride, indicative a of 95% yield.

Example 14—Optical Purification of (R)-Pramipexole Using Achiral AcidAddition

Pramipexole enantioenriched for the R(+) enantiomer (˜300 mg) wasdissolved in 10 ml of the chosen solvent at 75° C. (see examples inTable 8; ethanol or acetonitrile). Complete dissolution was observed inall samples. Acid addition was made at 1.05 molar equivalents for thep-TSA (solvent is ethanol; 2.97 ml of 0.5 M acid) and MSA (solvent isacetonitrile; 1.49 ml of 1.0 M acid), and 2.05 molar equivalents for thefumaric (solvent is acetonitrile; 5.84 ml of 0.5 M acid) and phosphoric(solvent is acetonitrile; 2.90 ml of 1.0 M acid). The reaction mixtureswere cooled to room temperature at a rate of 25° C./hour and stirred atroom temperature for an additional 19 hours. The solids obtained by thistrituration step were isolated by filtration and dried under high vacuumat room temperature. These products were analyzed by HPLC, ¹H NMR,thermal gravimetric analysis, differential scanning calorimetry, X-raypowder diffraction (XPRD), Fourier transform infrared spectroscopy andmoister-sorption analysis. The XPRD patterns showed that the p-TSA, MSAand fumarate salt forms of the (R)-pramipexole were crystalline, whilethe phosphate salt form of the (R)-pramipexole was amorphous.

Example 15—Industrial Scale Resolution of Racemic Diamine

A 72 L, unjacketed reactor was charged with racemic 2,6diamino-4,5,6,7-tetrahydro-benzothiazole (rac-diamine) (4.5 kg; 26.6mol) and 58.5 L water, and heated as a suspension to a temperature ofabout 60° C. to 65° C. Resolution of the enantiomers was achieved byaddition of one equivalent of (D)-(−)-Tartaric acid (3991 grams; 26.6mol) in 4.5 L of water, after which the resulting solution was heated toa temperature of about 70° C. to 75° C. and maintained at thistemperature for about 1 hour. The mixture was allowed to cool to atemperature of about 20° C. to 25° C. and stirred for an additional 15hours, after which the mixture was filtered and the solids were washed3× with water (6.3 L each wash).

The wet solids, which contain the R(+) enantiomer of the diamine, werecharged to the reactor followed by 54 L of water, and the mixture washeated to a temperature of about 70° C. to 75° C. for 2 hours. Themixture was allowed to cool to a temperature of about 20° C. to 25° C.and stirred for 17 hours. The mixture was then filtered and the solidswere washed 2× with water (4.5 L each wash). The wet solids weretransferred to a jacketed reactor and the reactor was charged with 8.1 Lof water. The mixture was cooled to a temperature of about 0° C. to 5°C. and cautiously charged with concentrated 1.625 L of HCl, followed by1.155 L of 50% NaOH to achieve a pH of about 9-10. During the additionthe temperature was maintained at about 0° C. to 5° C., and stirred foran additional hour at temperature. The resulting mixture was thenfiltered and the solids were washed 2× with cold (0° C. to 5° C.) water(1.125 L each wash). The solids were transferred to a jacketed reactorand were reslurried once more with 4.5 L of water at 0° C. to 5° C. Thesolids were filtered and dried under warm air (40° C. to 45° C.) to give1940 grams of the product (R(+) diamine) as a white solid, with an 86%yield for the R(+) enantiomer.

The mother liquors of the initial resolution step, which contain theS(−) enantiomer of the diamine, were concentrated to afford diamine witha 95.5% yield for the S(−) enantiomer.

TABLE 16 Experiments for industrial scale resolution of the R(+)enantiomer of diamine Input Yield (%) of Chemical Purity Chiral Purity(grams) R(+) enantiomer (AUC % by HPLC) (AUC % by HPLC) 1000 76 >99 98.34500 86 >99 98.5 4100 54 >99 98.5

Example 16—Industrial Scale Preparation of Propyl Tosylate

A 100 L glass, jacketed reactor was charged with 1-propanol (2.098 kg;34.9 mol), triethylamine (4.585 kg; 45.3 mol; 1.3 equivalents) and DCM(20.1 L). The mixture was cooled to a temperature of about 5° C. to 15°C. and cautiously charged with a solution of p-toluenesulfonyl chloride(6 kg; 31.47 mol; 0.9 equivalents) in DCM (10.5 L) over 30 minutes. Oncethe addition was complete, the mixture was warmed to a temperature ofabout 18° C. to 22° C. and stirred for 12 hours. The reaction mixturewas assayed by ¹H NMR (in CDCl₃) and deemed complete. HCl (6 N; 2.98 L)was cautiously charged while maintaining the temperature below 25° C.The aqueous phase was removed, and the organic phase was washed 2× withwater (21 L each wash), dried with MgSO₄, and filtered over Celite®. Thefiltered solids were then washed with DCM (4 L) and concentrated to aresidue. The residue was dissolved in heptane and concentrated again toafford a final propyl tosylate product (6.385 kg, 95% yield).

The present invention provides evidence that the dopamine receptoraffinity of (R)-pramipexole is actually much lower than previouslyappreciated. In a study using beagle dogs presented herein, it has beenshown that the functional separation between the (S)-pramipexole and(R)-pramipexole enantiomers (10,000-20,000 fold) is much greater thanpreviously expected. These data also show that contamination of thecomposition of pure (R)-pramipexole with small, known amounts of(S)-pramipexole results in a predictable shift in the MTD of thecomposition. These data demonstrate that (R)-pramipexole can be dosed atlevels that can more fully and unexpectedly exploit the lower-potencyneuroprotective potential of the compound without the theoretical MTDlimitation previously assumed, and without the need for dose titration.The application presents methods for using pure compositions of(R)-pramipexole in acute and chronic neurodegenerative disorderspreviously inaccessible to this drug and immediately at full-strengthwithout dose-titration and at higher theoretical MTDs. Additionally, thedata showing that a pure composition of (R)-pramipexole can be mixedwith a known amount of (S)-pramipexole to produce dopamine receptoragonist effects determined solely by the contribution of the(S)-enantiomer allows for the use of compositions comprising the mixtureof known amounts of pure (R)- and (S)-enantiomers for use inneurodegenerative disorders amenable to both dopamine receptor agonisttreatment and neuroprotection, such as PD.

Example 17—Industrial Scale Preparation of (R)-Pramipexole p-TSA Salt

Condition C: A 72 liter unjacketed reactor was charged with 1.84 kg(10.87 mol) of R(+)-2,6 diamino-4,5,6,7-tetrahydro-benzothiazole (R(+)diamine), followed by 14.7 L of dimethyl formamide (DMF). Undercontinuous stirring, the mixture was heated to a temperature of between65° C. and 68° C. A solution of 2926 grams propyl tosylate and 1761grams diisopropylethyleamine in 3.455 L DMF was added slowly over aperiod of 2 hours. The reaction was continued at 67° C. for anadditional 4 hours, after which the solution was gradually cooled toroom temperature (18° C. to 22° C.) and stirred for an additional 15hours. The solution was diluted with 14.72 L of MTBE over a time periodof 30 minutes, and stirred for an additional 1 hour. The precipitatedmaterial was collected by filtration and washed with 7.32 L MTBE,followed by 3 washes of 3.68 L each of ethanol, and a wash with 9.2 Lheptane. The washed precipitated cake was dried under high vacuum at 30°C. to 35° C. The final weight of the dried product was 2090 grams,representing a 50% yield.

Although the present invention has been described in considerable detailwith reference to certain preferred embodiments thereof, other versionsare possible. Therefore the spirit and scope of the appended claimsshould not be limited to the description and the preferred versionscontained within this specification.

1-23. (canceled)
 24. A method of treating amyotrophic lateral sclerosis(ALS) in a patient comprising: administering to the patient a singleunit dose of at least 150 milligrams of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine or apharmaceutically acceptable salt thereof; and administering adjunctivelyto the patient a therapeutically effective amount of riluzole.
 25. Themethod of claim 24, further comprising administering a mitochondrialenergy promoting agent is-selected from the group consisting ofresveratrol; creatine; erythropoietin; cholest-4-en-3-One, oxime(TRO-19622); and any combination thereof.
 26. (canceled)
 27. The methodof claim 24, wherein the steps of administering the single unit dose ofat least 150 milligrams of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine or apharmaceutically acceptable salt thereof and administering thetherapeutically effective amount of riluzole occurs concurrently. 28.The method of claim 24, wherein the steps of administering the singleunit dose of at least 150 milligrams of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine or apharmaceutically acceptable salt thereof and administering thetherapeutically effective amount of riluzole occurs separately.
 29. Themethod of claim 24, wherein the steps of administering the single unitdose of at least 150 milligrams of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine or apharmaceutically acceptable salt thereof and administering thetherapeutically effective amount of riluzole each individually occur oneor more times in a 24-hour period.
 30. The method of claim 24, whereinthe single unit dose of at least 150 milligrams of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine, orpharmaceutically acceptable salt thereof, has a 99.90% or greater chiralpurity.
 31. The method of claim 24, wherein the single unit dosecomprises at least about 200 mgs of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine or apharmaceutically acceptable salt thereof.
 32. The method of claim 24,wherein the single unit dose comprises at least about 300 mgs of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine or apharmaceutically acceptable salt thereof.
 33. The method of claim 24,wherein the single unit dose further comprises less than about 1.0 mg of(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine or apharmaceutically acceptable salt thereof.
 34. The method of claim 24,comprising a course of treatment wherein the steps of administering thesingle unit dose of at least 150 milligrams of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine or apharmaceutically acceptable salt thereof and administering thetherapeutically effective amount of riluzole is repeated one or moretimes in a 24-hour period for 5 days to one or more years.
 35. Themethod of claim 24, wherein the single unit dose of at least 150milligrams of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine or apharmaceutically acceptable salt thereof is a pharmaceuticallyacceptable salt of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine.
 36. Themethod of claim 35, wherein the pharmaceutically acceptable salt is(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diaminedihydrochloride monohydrate.
 37. A method of treating amyotrophiclateral sclerosis (ALS) in a patient comprising: administering to thepatient a single unit dose of about 150 milligrams to about 5,000milligrams of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine or apharmaceutically acceptable salt thereof in a pharmaceuticalcomposition; and administering adjunctively to the patient atherapeutically effective amount of riluzole.
 38. The method of claim37, further comprising administering a mitochondrial energy promotingagent selected from the group consisting of resveratrol; creatine;erythropoietin; cholest-4-en-3-One, oxime (TRO-19622); and anycombination thereof.